Medicinal Natural Products: A Biosynthetic Approach, Third Edition

Author: Paul M. Dewick

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Medicinal Natural Products

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

Medicinal Natural Products A Biosynthetic Approach 3rd Edition

Paul M Dewick formerly University of Nottingham, UK

A John Wiley and Sons, Ltd., Publication

This edition first published 2009 © 2009 John Wiley & Sons Ltd Registered office John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, United Kingdom For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com. The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. This work is sold with the understanding that the publisher is not engaged in rendering professional services. The advice and strategies contained herein may not be suitable for every situation. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of experimental reagents, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each chemical, piece of equipment, reagent, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Dewick, Paul M. Medicinal natural products : a biosynthetic approach / Paul M. Dewick. – 3rd ed. p. cm. Includes bibliographical references and index. ISBN 978-0-470-74168-9 (cloth) – ISBN 978-0-470-74167-2 (pbk. : alk. paper) 1. Natural products. 2. Biosynthesis. 3. Pharmacognosy. I. Title. RS160.D48 2008 615 .3 – dc22 2008044502 A catalogue record for this book is available from the British Library. ISBN: 978-0-470-74168-9 (HB) 978-0-470-74167-2 (PB) Typeset in 9pt/11pt Times Roman by Laserwords Private Limited, Chennai, India Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wiltshire Cover photograph English yew (Taxus baccata). Photograph: Paul Dewick

for Jane

CONTENTS

1 About this book, and how to use it . . . The subject . . . . . . . . . . . . . The aim . . . . . . . . . . . . . . The approach . . . . . . . . . . . . The topics . . . . . . . . . . . . . The figures . . . . . . . . . . . . . Further reading . . . . . . . . . . . . What to study . . . . . . . . . . . . What to learn . . . . . . . . . . . . Nomenclature . . . . . . . . . . . . Conventions regarding acids, bases, and ions Some common abbreviations . . . . . . Further reading . . . . . . . . . . . .

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2 Secondary metabolism: the building blocks and construction Primary and secondary metabolism . . . . . . . . . . . The building blocks . . . . . . . . . . . . . . . . . The construction mechanisms . . . . . . . . . . . . . Alkylation reactions: nucleophilic substitution . . . . Alkylation reactions: electrophilic addition . . . . . Wagner–Meerwein rearrangements . . . . . . . . Aldol and Claisen reactions . . . . . . . . . . . Imine formation and the Mannich reaction . . . . . Amino acids and transamination . . . . . . . . . Decarboxylation reactions . . . . . . . . . . . . Oxidation and reduction reactions . . . . . . . . . Dehydrogenases . . . . . . . . . . . . . Oxidases . . . . . . . . . . . . . . . . Monooxygenases . . . . . . . . . . . . Dioxygenases . . . . . . . . . . . . . . Amine oxidases . . . . . . . . . . . . . Baeyer–Villiger monooxygenases . . . . . . Phenolic oxidative coupling . . . . . . . . . . . Halogenation reactions . . . . . . . . . . . . . Glycosylation reactions . . . . . . . . . . . . . Elucidating biosynthetic pathways . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . . . .

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mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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viii Contents 3 The acetate pathway: fatty acids and polyketides . Fatty acid synthase: saturated fatty acids . . . . . . Unsaturated fatty acids . . . . . . . . . . . . . Uncommon fatty acids . . . . . . . . . . . . . Prostaglandins . . . . . . . . . . . . . . . . Thromboxanes . . . . . . . . . . . . . . . . Leukotrienes . . . . . . . . . . . . . . . . . Polyketide synthases: generalities . . . . . . . . . Polyketide synthases: macrolides . . . . . . . . . Polyketide synthases: linear polyketides and polyethers Diels–Alder cyclizations . . . . . . . . . . . . Polyketide synthases: aromatics . . . . . . . . . Cyclizations . . . . . . . . . . . . . . Post-polyketide synthase modifications . . . . Starter groups . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . 4 The shikimate pathway: aromatic amino acids and Aromatic amino acids and simple benzoic acids . . Phenylpropanoids . . . . . . . . . . . . . . Cinnamic acids and esters . . . . . . . . Lignans and lignin . . . . . . . . . . . Phenylpropenes . . . . . . . . . . . . Benzoic acids from C6 C3 compounds . . . Coumarins . . . . . . . . . . . . . . Aromatic polyketides . . . . . . . . . . . . Styrylpyrones, diarylheptanoids . . . . . . Flavonoids and stilbenes . . . . . . . . Flavonolignans . . . . . . . . . . . . Isoflavonoids . . . . . . . . . . . . . Terpenoid quinones . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . 5 The mevalonate and methylerythritol phosphate Mevalonic acid and methylerythritol phosphate . Hemiterpenes (C5 ) . . . . . . . . . . . . Monoterpenes (C10 ) . . . . . . . . . . . . Irregular monoterpenes . . . . . . . . . . . Iridoids (C10 ) . . . . . . . . . . . . . . Sesquiterpenes (C15 ) . . . . . . . . . . . . Diterpenes (C20 ) . . . . . . . . . . . . . Sesterterpenes (C25 ) . . . . . . . . . . . . Triterpenes (C30 ) . . . . . . . . . . . . . Triterpenoid saponins . . . . . . . . Steroids . . . . . . . . . . . . . . . . Stereochemistry and nomenclature . . . Cholesterol . . . . . . . . . . . . Phytosterols . . . . . . . . . . . . Vitamin D . . . . . . . . . . . . . Steroidal saponins . . . . . . . . . . Cardioactive glycosides . . . . . . . . Bile acids . . . . . . . . . . . . .

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39 . 39 . 44 . 53 . 58 . 64 . 64 . 66 . 68 . 90 . 96 . 96 . 99 . 103 . 116 . 131

phenylpropanoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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137 137 148 148 152 156 157 161 166 166 167 173 174 178 184

steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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187 188 192 193 204 206 210 223 234 234 242 247 247 248 251 256 259 265 275

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pathways: terpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents ix Adrenocortical hormones/corticosteroids Semi-synthesis of corticosteroids . . . Progestogens . . . . . . . . . . . Oestrogens . . . . . . . . . . . . Androgens . . . . . . . . . . . . Tetraterpenes (C40 ) . . . . . . . . . . . Higher terpenoids . . . . . . . . . . . . Further reading . . . . . . . . . . . . .

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277 277 287 290 296 298 306 306

6 Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . Alkaloids derived from ornithine . . . . . . . . . . . . . . . Polyamines . . . . . . . . . . . . . . . . . . . . Pyrrolidine and tropane alkaloids . . . . . . . . . . . . Pyrrolizidine alkaloids . . . . . . . . . . . . . . . . Alkaloids derived from lysine . . . . . . . . . . . . . . . . Piperidine alkaloids . . . . . . . . . . . . . . . . . Quinolizidine alkaloids . . . . . . . . . . . . . . . . Indolizidine alkaloids . . . . . . . . . . . . . . . . Alkaloids derived from nicotinic acid . . . . . . . . . . . . . Pyridine alkaloids . . . . . . . . . . . . . . . . . . Alkaloids derived from tyrosine . . . . . . . . . . . . . . . Phenylethylamines and simple tetrahydroisoquinoline alkaloids Modified benzyltetrahydroisoquinoline alkaloids . . . . . . Phenethylisoquinoline alkaloids . . . . . . . . . . . . . Terpenoid tetrahydroisoquinoline alkaloids . . . . . . . . Amaryllidaceae alkaloids . . . . . . . . . . . . . . . Alkaloids derived from tryptophan . . . . . . . . . . . . . . Simple indole alkaloids . . . . . . . . . . . . . . . . Simple β-carboline alkaloids . . . . . . . . . . . . . . Terpenoid indole alkaloids . . . . . . . . . . . . . . . Quinoline alkaloids . . . . . . . . . . . . . . . . . Pyrroloindole alkaloids . . . . . . . . . . . . . . . . Ergot alkaloids . . . . . . . . . . . . . . . . . . . Alkaloids derived from anthranilic acid . . . . . . . . . . . . Quinazoline alkaloids . . . . . . . . . . . . . . . . Quinoline and acridine alkaloids . . . . . . . . . . . . Alkaloids derived from histidine . . . . . . . . . . . . . . . Imidazole alkaloids . . . . . . . . . . . . . . . . . Alkaloids derived by amination reactions . . . . . . . . . . . . Acetate-derived alkaloids . . . . . . . . . . . . . . . Phenylalanine-derived alkaloids . . . . . . . . . . . . . Terpenoid alkaloids . . . . . . . . . . . . . . . . . Steroidal alkaloids . . . . . . . . . . . . . . . . . . Purine alkaloids . . . . . . . . . . . . . . . . . . . . . Caffeine . . . . . . . . . . . . . . . . . . . . . . Saxitoxin and tetrodotoxin . . . . . . . . . . . . . . . Further reading . . . . . . . . . . . . . . . . . . . . . .

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311 311 311 312 324 326 326 328 330 331 331 336 336 346 359 363 365 366 366 369 369 380 385 387 395 395 396 398 398 400 401 401 406 406 413 413 416 417

7 Peptides, proteins, and other amino Peptides and proteins . . . . . . Ribosomal peptide biosynthesis . . Peptide hormones . . . . . . . .

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derivatives . . . . . . . . . . . . . . .

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x Contents Thyroid hormones . . . . . . . . Hypothalamic hormones . . . . . Anterior pituitary hormones . . . . Posterior pituitary hormones . . . . Pancreatic hormones . . . . . . . Interferons . . . . . . . . . . . Opioid peptides . . . . . . . . . Ribosomal peptide toxins . . . . . . . . Enzymes . . . . . . . . . . . . . . Non-ribosomal peptide biosynthesis . . . . Modified peptides: penicillins, cephalosporins, Penicillins . . . . . . . . . . . Cephalosporins . . . . . . . . . Other β-lactams . . . . . . . . . Cyanogenic glycosides . . . . . . . . . Glucosinolates . . . . . . . . . . . . Cysteine sulfoxides . . . . . . . . . . Further reading . . . . . . . . . . . . 8 Carbohydrates Monosaccharides Oligosaccharides Polysaccharides Aminosugars and Further reading . Index

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426 427 429 430 432 433 434 434 438 438 458 458 465 469 476 477 480 481

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1

ABOUT THIS BOOK, AND HOW TO USE IT

THE SUBJECT This book has been written primarily for pharmacy students to provide a modern text to complement lecture courses dealing with pharmacognosy and the use of natural products in medicine. Nevertheless, it should be of value in other courses where the study of natural products is included, although the examples chosen are predominantly those possessing pharmacological activity. For centuries, drugs were entirely of natural origin and composed of herbs, animal products, and inorganic materials. Early remedies may have combined these ingredients with witchcraft, mysticism, astrology, or religion, but it is certain that those treatments that were effective were subsequently recorded and documented, thus leading to the early Herbals. The science of pharmacognosy – the knowledge of drugs – grew from these records to provide a disciplined, scientific description of natural materials used in medicine. Herbs formed the bulk of these remedies. As chemical techniques improved, the active constituents were isolated from plants, were structurally characterized, and, in due course, many were synthesized in the laboratory. Sometimes, more active, better-tolerated drugs were produced by chemical modifications (semi-synthesis), or by total synthesis of analogues of the active principles. Gradually, synthetic compounds superseded many of the old plant drugs, though certain plant-derived agents were never surpassed and remain as valued medicines to this day. Natural drugs derived from microorganisms have a much shorter history, and their major impact on medicine goes back only about 60 years to the introduction of the antibiotic penicillin. Microbially produced

antibiotics now account for a very high proportion of the drugs commonly prescribed. There is currently a renewed interest in pharmacologically active natural products, be they from plants, microorganisms, or animals, terrestrial or marine, in the continued search for new drugs, particularly for disease states where our present range of drugs is less effective than we would wish. This is being reflected in a growing number of natural products or natural-product-inspired drugs entering medicine. Herbal remedies are also enjoying a revival as many sufferers turn away from modern drugs and embrace ‘complementary medicine’. THE AIM Many university pharmacy courses include a pharmacognosy component covering a study of plant-derived drugs; traditionally, this area of natural products has been taught separately from the microbially derived antibiotics, or the animal-related steroidal and prostanoid drugs. Such topics have usually formed part of a pharmaceutical chemistry course. The traditional boundaries may still remain, despite a general change in pharmacognosy teaching from a descriptive study to a phytochemical-based approach, a trend towards integrating pharmacognosy within pharmaceutical chemistry, and the general adoption of modular course structures. A chemistry-based teaching programme encompassing all types of natural products of medicinal importance, semi-synthetic derivatives, and synthetic analogues based on natural product templates is a logical development. This book provides a suitable text to complement such a programme, and attempts to break down the artificial divisions.

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

2 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition THE APPROACH This book provides a groundwork in natural product chemistry/phytochemistry by considering biosynthesis – the metabolic sequences leading to various selected classes of natural products. This allows application of fundamental chemical principles and displays the relationships between the diverse structures encountered in nature, thus providing a rationale for natural products and replacing a descriptive approach with one based more on deductive reasoning. It also helps to transform complicated structures into a comprehensible combination of simpler fragments; natural product structures can be quite complex. Subdivision of the topics is predominantly via biosynthesis, not by class or activity, and this provides a logical sequence of structural types and avoids a catalogue effect. There is extensive use of chemical schemes and mechanism, with detailed mechanistic explanations being annotated to the schemes, as well as outline discussions in the text. Lots of cross-referencing is included to emphasize links and similarities; it is not necessary to follow these to understand the current material, but they are used to stress that the concept has been met before, or that other uses will be met in due course. As important classes of compounds or drugs are reached, more detailed information is then provided in the form of short separate monographs in boxes, which can be studied or omitted as required, in the latter case allowing the main theme to continue. The monograph information covers sources, production methods, principal components, drug use, mode of action, semi-synthetic derivatives, synthetic analogues, etc., as appropriate. Those materials currently employed as drugs, or being tested clinically, are emphasized in the monographs by the use of bold type. THE TOPICS A preliminary chapter is used to outline the main building blocks, the basic construction mechanisms employed in the biosynthesis of natural products, and how metabolic pathways are deduced. Most of the fundamental principles should be familiar and will have been met previously in courses dealing with the basics of organic chemistry and biochemistry. These principles are then seen in action as representative natural product structures are described in the following chapters. The topics selected are subdivided initially into areas of metabolism fed by the acetate, shikimate, mevalonate, and methylerythritol phosphate pathways. The remaining chapters then cover alkaloids, peptides and proteins, and carbohydrates. Not all classes of natural products can be covered, and the book is intended as an introductory text, not a comprehensive reference work.

The book tries to include a high proportion of those natural products currently used in medicine, the major drugs that are derived from natural materials by semi-synthesis, and those drugs which are structural analogues. Some of the compounds mentioned may have a significant biological activity which is of interest, but not medicinally useful. The book is also designed to be forward looking and gives information on possible leads to new drugs and materials in clinical trials. THE FIGURES A cursory glance through the book will show that a considerable portion of the content is in the form of chemical structures and schemes. The schemes and figures are used to provide maximum information as concisely as possible. The following guidelines should be appreciated: • A figure may present a composite scheme derived from

studies in more than one organism. • Comments in italics provide an explanation in chemical

•

•

•

•

•

•

terms for the biochemical reaction; detailed enzymic mechanisms are not usually considered. Schemes in separate frames show a mechanism for part of the sequence, the derivation of a substrate, or perhaps structurally related systems. Although enzymic reactions may be reversible, single rather than reversible arrows are used, unless the transformation is one that may be implicated in both directions, e.g. amino acid/keto acid transaminations. E1, E2, etc., refer to enzymes catalysing the transformation, when known. Where no enzyme is indicated, the transformation may well have been determined by other methodology, e.g. isotope tracer studies. Speculative conversions may be included, but are clearly indicated. Enzyme names shown are the commonly accepted names; in general, only one name is given, even though alternative names may also be in current use. Proteins identified via the corresponding gene are often assigned a code name/number by researchers, and no systematic name has been proposed. This means that proteins carrying out the same transformation in different organisms may be assigned different codes. Double-headed curly arrows are used to represent an addition–elimination mechanism as follows: addition–elimination O

O

L

Nu shorthand style Nu

Nu O L

O L

Nu

L

About This Book, and How to Use It 3 FURTHER READING A selection of articles suitable for supplementary reading is provided at the end of each chapter. In general, these are not chosen from the primary literature, but are recent review articles covering broader aspects of the topic. They are also located in easily accessible journals rather than books, and have been chosen as the most student friendly. In certain cases, the most recent reviews available may be somewhat less up to date than the information covered in this book. All of the selected articles contain information considered appropriate to this book, e.g. reviews on ‘synthesis’ may contain sections on structural aspects, biosynthesis, or pharmacology. WHAT TO STUDY Coverage is fairly extensive to allow maximum flexibility for courses in different institutions, and not all of the material will be required for any one course. However, because of the many subdivisions and the highlighted keywords, it should be relatively easy to find and select the material appropriate for a particular course. On the other hand, the detail given in monographs is purposely limited to ensure students are provided with enough factual information, but are not faced with the need to assess whether or not the material is relevant. Even so, these monographs will undoubtedly contain data which exceed the scope of any individual course. It is thus necessary to apply selectivity, and portions of the book will be surplus to immediate requirements. The book is designed to be user friendly, suitable for modular courses and student-centred learning exercises, and a starting point for later project and dissertation work. The information presented is as up to date as possible; undoubtedly, new research will be published that modifies or even contradicts some of the statements made. The reader is asked always to be critical and to maintain a degree of flexibility when reading the scientific literature, and to appreciate that science is always changing. WHAT TO LEARN The primary aim of the book is not to rely just on factual information, but to impart an understanding of natural product structures and the way they are put together by living organisms. Rationalization based on mechanistic reasoning is paramount. The sequences themselves are not important, whilst the names of chemicals and the enzymes involved in the pathways are even less relevant and included only for information; it is the mechanistic explanations that are the essence. Students should concentrate on understanding the broad features of the

sequences and absorb sufficient information to be able to predict how and why intermediates might be elaborated and transformed. The mechanistic explanations appended to the schemes should reinforce this approach. Anyone who commits to memory a sequence of reactions for examination purposes has missed the point. There is no alternative to memory for some of the material covered in the monographs, if it is required; wherever possible, information should be reduced to a concept that can be deduced, rather than remembered. The approach used here should help students to develop such deductive skills.

NOMENCLATURE Natural product structures are usually quite complex, some exceedingly so, and fully systematic nomenclature becomes impracticable. Names are thus typically based on so-called trivial nomenclature, in which the discoverer of the natural product exerts their right to name the compound. The organism in which the compound is found is frequently chosen to supply the root name, e.g. hyoscyamine from Hyoscyamus, atropine from Atropa, or penicillin from Penicillium. Name suffixes might be -in to indicate ‘a constituent of’, -oside to show the compound is a sugar derivative, -genin for the aglycone released by hydrolysis of the sugar derivative, -toxin for a poisonous constituent, or may reflect chemical functionality, such as -one or -ol. Traditionally, -ine is always used for alkaloids (amines). Structurally related compounds are then named as derivatives of the original, using standard prefixes, such as hydroxy-, methoxy-, methyl-, dihydro-, homo-, etc. for added substituents, or deoxy-, demethyl-, demethoxy-, dehydro-, nor-, etc. for removed substituents. Homo- is used to indicate one carbon more, whereas nor- means one carbon less. The position of this change is then indicated by systematic numbering of the carbon chains or rings. Some groups of compounds, such as steroids, fatty acids, and prostaglandins, are named semi-systematically from an accepted root name for the complex hydrocarbon skeleton. In this book, almost all structures depicted in the figures carry a name; this is primarily to help identification, and, for the student, structural features should be regarded as more pertinent than the names used. It will soon become apparent that drug names chosen by pharmaceutical manufacturers are quite random, and in most cases have no particular relationship to the chemical structure. However, some common stems are employed to indicate relationship to a group of therapeutically active drugs. Examples are -cillin for antibiotics of the penicillin group, cef- for antibiotics of the cephalosporin group, -mycin for antibiotics produced

4 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition by Streptomyces, -caine for local anaesthetics, -stat for enzyme inhibitors, -vastatin for HMGCoA reductase inhibitors, prost for prostaglandins, and gest for progestogens. We are also currently still in a transitional period during which many established drug names are being changed to recommended international non-proprietary names (rINNs); both names are included here, with the rINN preceding the older name. CONVENTIONS REGARDING ACIDS, BASES, AND IONS In many structures, the abbreviation OP is used to represent the phosphate group and OPP the diphosphate (or pyrophosphate) group: O

O RO

P

RO

O

P

O O

O

O

P

O

Fru GABA Gal GFPP

O

a diphosphate (pyrophosphate) ROPP

At physiological pH values, these groups will be ionized as shown, but in schemes where structures are given in full, the non-ionized acids are usually depicted. This is done primarily to simplify structures, to eliminate the need for counter-ions, and to avoid mechanistic confusion. Likewise, amino acids are shown in non-ionized form, although they will typically exist as zwitterions: CO2H

H NH2 L-amino acid

R

CO2

H NH3 zwitterion

Ionized and non-ionized forms of many compounds are regarded as synonymous in the text; thus, acetate/acetic acid, shikimate/shikimic acid, and mevalonate/mevalonic acid may be used according to the author’s whim and context and have no especial relevance. SOME COMMON ABBREVIATIONS 5-HT ACP ADP Ara ATP B: CoA

DXP Enz FAD FADH2 FAS FH4 FMN FMNH2 FPP

GGPP

a phosphate ROP

R

Dig DMAPP

5-hydroxytryptamine acyl carrier protein adenosine diphosphate arabinose adenosine triphosphate general base coenzyme A as part of a thioester, e.g. acetyl-CoA (CH3 COSCoA)

Glc GPP HA HSCoA IPP LT Mann MEP MVA NAD+ NADH NADP+ NADPH NRPS O P P PCP PEP PG PKS PLP PP PP Rha Rib

digitoxose dimethylallyl diphosphate (dimethylallyl pyrophosphate) 1-deoxyxylulose 5-phosphate enzyme (usually shown as thiol: EnzSH) flavin adenine dinucleotide flavin adenine dinucleotide (reduced) fatty acid synthase tetrahydrofolic acid flavin mononucleotide flavin mononucleotide (reduced) farnesyl diphosphate (farnesyl pyrophosphate) fructose γ-aminobutyric acid galactose geranylfarnesyl diphosphate (geranylfarnesyl pyrophosphate) geranylgeranyl diphosphate (geranylgeranyl pyrophosphate) glucose geranyl diphosphate (geranyl pyrophosphate) general acid coenzyme A isopentenyl diphosphate (isopentenyl pyrophosphate) leukotriene mannose methylerythritol phosphate mevalonic acid nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide phosphate nicotinamide adenine dinucleotide phosphate (reduced) non-ribosomal peptide synthase oxidation – in schemes phosphate – in text phosphate – in structures peptidyl carrier protein phosphoenolpyruvate prostaglandin polyketide synthase pyridoxal 5 -phosphate diphosphate (pyrophosphate) – in text diphosphate (pyrophosphate) – in structures rhamnose ribose

About This Book, and How to Use It 5 SAM TPP TX UDP UDPGlc UTP W–M Xyl hν

S-adenosyl methionine thiamine diphosphate (thiamine pyrophosphate) thromboxane uridine diphosphate uridine diphosphoglucose uridine triphosphate Wagner–Meerwein (rearrangement) xylose heat electromagnetic radiation; usually UV or visible

FURTHER READING Pharmacognosy, Phytochemistry, Natural Drugs Books Bruneton J (1999) Pharmacognosy, Phytochemistry and Medicinal Plants. Lavoisier, Paris. Evans WC (2001) Trease & Evans’ Pharmacognosy. Saunders, London. Heinrich M, Barnes J, Gibbons S and Williamson EM (2004) Fundamentals of Pharmacognosy and Phytotherapy. Churchill Livingstone, London. Samuelsson G (2004) Drugs of Natural Origin. A Textbook of Pharmacognosy. Swedish Pharmaceutical Press, Stockholm.

Reviews Baker DD, Chu M, Oza U and Rajgarhia V (2007) The value of natural products to future pharmaceutical discovery. Nat Prod Rep 24, 1225–1244.

Bode HB and M¨uller R (2005) The impact of bacterial genomics on natural product research. Angew Chem Int Ed 44, 6828–6846. Butler MS (2004) The role of natural product chemistry in drug discovery. J Nat Prod 67, 2141–2153; errata (2006) 69, 172. Butler MS (2008) Natural products to drugs: natural product-derived compounds in clinical trials. Nat Prod Rep 25, 475–516. Chin Y-W, Balunas MJ, Chai HB and Kinghorn AD (2006) Drug discovery from natural sources. AAPS J 8, 239–253. Esp´ın JC, Garc´ıa-Conesa MT and Tom´as-Barber´an FA (2007) Nutraceuticals: facts and fiction. Phytochemistry 68, 2986–3008. Kennedy J (2008) Mutasynthesis, chemobiosynthesis, and back to semi-synthesis: combining synthetic chemistry and biosynthetic engineering for diversifying natural products. Nat Prod Rep 25, 25–34. McChesney JD, Venkataraman SK and Henri JT (2007) Plant natural products: back to the future or into extinction? Phytochemistry 68, 2015–2022. Misiek M and Hoffmeister D (2007) Fungal genetics, genomics, and secondary metabolites in pharmaceutical sciences. Planta Med 73, 103–115. Newman DJ (2008) Natural products as leads to potential drugs: an old process or the new hope for drug discovery? J Med Chem 51, 2589–2599. Newman DJ and Cragg GM (2004) Marine natural products and related compounds in clinical and advanced preclinical trials. J Nat Prod 67, 1216–1238. Newman DJ and Cragg GM (2007) Natural products as sources of new drugs over the last 25 years. J Nat Prod 70, 461–477. Phillipson JD (2007) Phytochemistry and pharmacognosy. Phytochemistry 68, 2960–2972. Tietze LF, Bell HP and Chandrasekhar S (2003) Natural product hybrids as new leads for drug discovery. Angew Chem Int Ed 42, 3996–4028.

2

SECONDARY METABOLISM: THE BUILDING BLOCKS AND CONSTRUCTION MECHANISMS

PRIMARY AND SECONDARY METABOLISM All organisms need to transform and interconvert a vast number of organic compounds to enable them to live, grow, and reproduce. They need to provide themselves with energy in the form of ATP, and a supply of building blocks to construct their own tissues. An integrated network of enzyme-mediated and carefully regulated chemical reactions is used for this purpose, collectively referred to as intermediary metabolism, and the pathways involved are termed metabolic pathways. Some of the crucially important molecules of life are carbohydrates, proteins, fats, and nucleic acids. Apart from fats, these tend to be polymeric materials. Carbohydrates are composed of sugar units, whilst proteins are made up from amino acids, and nucleic acids are based on nucleotides. Organisms vary widely in their capacity to synthesize and transform chemicals. For instance, plants are very efficient at synthesizing organic compounds via photosynthesis from inorganic materials found in the environment, whilst other organisms, such as animals and microorganisms, rely on obtaining their raw materials in their diet, e.g. by consuming plants. Thus, many of the metabolic pathways are concerned with degrading materials taken in as food, whilst others are then required to synthesize specialized molecules from the basic compounds so obtained. Despite the extremely varied characteristics of living organisms, the pathways for generally modifying and synthesizing carbohydrates, proteins, fats, and nucleic acids are found to be essentially the same in all organisms, apart from minor variations. These

processes demonstrate the fundamental unity of all living matter, and are collectively described as primary metabolism, with the compounds involved in the pathways being termed primary metabolites. Thus, degradation of carbohydrates and sugars generally proceeds via the well-characterized pathways known as glycolysis and the Krebs/citric acid/tricarboxylic acid cycle, which release energy from the organic compounds by oxidative reactions. Oxidation of fatty acids from fats by the sequence called β-oxidation also provides energy. Aerobic organisms are able to optimize these processes by adding on a further process, namely oxidative phosphorylation. This improves the efficiency of oxidation by incorporating a more general process applicable to the oxidation of a wide variety of substrates rather than having to provide specific processes for each individual substrate. Proteins taken in via the diet provide amino acids, but the proportions of each will almost certainly vary from the organism’s requirements. Metabolic pathways are thus available to interconvert amino acids, or degrade those not required and thus provide a further source of energy. Most organisms can synthesize only a proportion of the amino acids they actually require for protein synthesis. Those structures not synthesized, so-called essential amino acids, must be obtained from external sources. In contrast to these primary metabolic pathways, which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with compounds which have a much more limited distribution in nature. Such compounds, called secondary metabolites,

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

8 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition are found in only specific organisms, or groups of organisms, and are an expression of the individuality of species. Secondary metabolites are not necessarily produced under all conditions, and in the vast majority of cases the function of these compounds and their benefit to the organism are not yet known. Some are undoubtedly produced for easily appreciated reasons, e.g. as toxic materials providing defence against predators, as volatile attractants towards the same or other species, or as colouring agents to attract or warn other species, but it is logical to assume that all do play some vital role for the well-being of the producer. It is this area of secondary metabolism which provides most of the pharmacologically active natural products. It is thus fairly obvious that the human diet could be both unpalatable and remarkably dangerous if all plants, animals, and fungi produced the same range of compounds. The above generalizations distinguishing primary and secondary metabolites unfortunately leave a ‘grey area’ at the boundary, so that some groups of natural products could be assigned to either division. Fatty acids and sugars provide good examples, in that most are best described as primary metabolites, whilst some representatives are extremely rare and found only in a handful of species. Likewise, steroid biosynthesis produces a range of widely distributed fundamental structures, yet some steroids, many of them with pronounced pharmacological activity, are restricted to certain organisms. Hopefully, the blurring of the boundaries will not cause confusion; the subdivision into primary metabolism (≡ biochemistry) or secondary metabolism (≡ natural products chemistry) is merely a convenience and there is considerable overlap.

THE BUILDING BLOCKS The building blocks for secondary metabolites are derived from primary metabolism as indicated in Figure 2.1. This scheme outlines how metabolites from the fundamental processes of photosynthesis, glycolysis, and the Krebs cycle are tapped off from energy-generating processes to provide biosynthetic intermediates. The number of building blocks needed is surprisingly few, and as with any child’s construction set, a vast array of objects can be built up from a limited number of basic building blocks. By far the most important building blocks employed in the biosynthesis of secondary metabolites are derived from the intermediates acetyl coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid, and methylerythritol phosphate. These are utilized respectively in the acetate, shikimate, mevalonate, and methylerythritol phosphate pathways, which form the basis of succeeding chapters. Acetyl-CoA is formed by oxidative decarboxylation of

the glycolytic pathway product pyruvic acid. It is also produced by the β-oxidation of fatty acids, effectively reversing the process by which fatty acids are themselves synthesized from acetyl-CoA. Important secondary metabolites formed from the acetate pathway include phenols, prostaglandins, and macrolide antibiotics, together with various fatty acids and derivatives at the primary–secondary metabolism interface. Shikimic acid is produced from a combination of phosphoenolpyruvate, a glycolytic pathway intermediate, and erythrose 4-phosphate from the pentose phosphate pathway. The reactions of the pentose phosphate cycle may be employed for the degradation of glucose, but they also feature in the synthesis of sugars by photosynthesis. The shikimate pathway leads to a variety of phenols, cinnamic acid derivatives, lignans, and alkaloids. Mevalonic acid is itself formed from three molecules of acetyl-CoA, but the mevalonate pathway channels acetate into a different series of compounds than does the acetate pathway. Methylerythritol phosphate arises from a combination of two glycolytic pathway intermediates, namely pyruvic acid and glyceraldehyde 3-phosphate by way of deoxyxylulose phosphate. The mevalonate and methylerythritol phosphate pathways are together responsible for the biosynthesis of a vast array of terpenoid and steroid metabolites. In addition to acetyl-CoA, shikimic acid, mevalonic acid, and methylerythritol phosphate, other building blocks based on amino acids are frequently employed in natural product synthesis. Peptides, proteins, alkaloids, and many antibiotics are derived from amino acids, and the origins of some of the more important amino acid components of these are briefly indicated in Figure 2.1. Intermediates from the glycolytic pathway and the Krebs cycle are used in constructing many of them, but the aromatic amino acids phenylalanine, tyrosine, and tryptophan are themselves products from the shikimate pathway. Ornithine, an amino acid not found in proteins, and its homologue lysine, are important alkaloid precursors and have their origins in Krebs cycle intermediates. Of special significance is the appreciation that secondary metabolites can be synthesized by combining several building blocks of the same type, or by using a mixture of different building blocks. This expands structural diversity and, consequently, makes subdivisions based entirely on biosynthetic pathways rather more difficult. A typical natural product might be produced by combining elements from the acetate, shikimate, and methylerythritol phosphate pathways, for example. Many secondary metabolites also contain one or more sugar units in their structure, either simple primary metabolites,

Secondary Metabolism: The Building Blocks and Construction Mechanisms

9

GLYCOLYSIS OH

OH OH

O HO

O

PO

PO

OH

PENTOSE PHOSPHATE CYCLE OH

OH

OH

OH

OH

D-glucose

erythrose 4-P

glucose 6-P

OHC

CO2H

PHOTOSYNTHESIS

O

OH

OH CO2H

NH2

PO NH2

glyceraldehyde 3-P

glycine

L-phenylalanine

CO2H CO2H

HS

HO2C

CO2H

HO

NH2

NH2

HO

PO

L-serine

L-cysteine

CO2H

OH OH

3-phosphoglyceric acid

NH2

HO

L-tyrosine

OH

CO2H

SHIKIMIC ACID HO2C

CO2H

NH2

OP NH

NH2

L-tryptophan

phosphoenolpyruvate

L-valine

OH

OH

CO2H

HO2C

O

L-alanine

OP

OP

O

NH2

pyruvic acid

OH

OH

deoxyxylulose 5-P

OH

METHYLERYTHRITOL 4-P OH

CO2H

CoAS

O

NH2

HO2C

L-leucine

OH

ACETYL-CoA

MEVALONIC ACID KREBS CYCLE CO2H

HO2C

NH2

CO2H NH2

HO2C

O

L-aspartic acid

L-isoleucine

CO2H

HO2C

CO2H O

oxaloacetic acid

2-oxoglutaric acid

HO2C

CO2H NH2

L-glutamic acid

NH S

CO2H NH2

L-methionine

H2N

CO2H NH2

L-lysine

H2N

CO2H

N H

L-arginine

NH2

CO2H

H2N

NH2

L-ornithine

Figure 2.1 such as glucose or ribose, or alternatively substantially modified and unusual sugars. To appreciate how a natural product is elaborated, it is of value to be able to dissect its structure into the basic building blocks from which it is made up and to use fundamental chemical mechanisms to propose how these are joined together. With a little experience and practice, this becomes a relatively simple

process and it allows the molecule to be rationalized, thus exposing logical relationships between apparently quite different structures. In this way, similarities become much more meaningful than differences, and an understanding of biosynthetic pathways allows rational connecting links to be established. This forms the basic approach in this book.

10 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Relatively few building blocks are routinely employed, and the following list, though not comprehensive, includes those most frequently encountered in producing the carbon and nitrogen skeleton of a natural product. As we shall see, oxygen atoms can be introduced and removed by a variety of processes, and so are not considered in the initial analysis, except as a pointer to an acetate (see page 101) or shikimate (see page 140) origin. The structural features of these building blocks are shown in Figure 2.2. • C1 : The simplest of the building blocks is composed of

a single carbon atom, usually in the form of a methyl group, and most frequently it is attached to oxygen

or nitrogen, but occasionally to carbon or sulfur. It is derived from the S-methyl of l-methionine. The methylenedioxy group (–OCH2 O–) is also an example of a C1 unit. • C2 : A two-carbon unit may be supplied by acetyl-CoA. This could be a simple acetyl group, as in an ester, but more frequently it forms part of a long alkyl chain (as in a fatty acid) or may be part of an aromatic system (e.g. phenols). Of particular relevance is that, in the latter examples, acetyl-CoA is first converted into the more reactive malonyl-CoA before its incorporation. • C5 : The branched-chain C5 ‘isoprene’ unit is a feature of compounds formed from mevalonate or

The building blocks H3C

S

CO2H L-Met

X CH3

NH2

(X = O, N, S, C)

C1 CO2H

SCoA

SCoA

O

O

acetyl-CoA

C

C

C2

malonyl-CoA

HO

SCoA HO2C

3× O acetyl-CoA

OH mevalonic acid

OH

HO OP

OP

isoprene unit C5

OH OH methylerythritol phosphate

O OH deoxyxylulose phosphate CO2H NH2 L-Phe

C6C2 CO2H

HO

NH2

C6C3

L-Tyr

C6C1 Figure 2.2

Secondary Metabolism: The Building Blocks and Construction Mechanisms CO2H

11

CO2H

NH2 NH2

L-Phe N

N

NH

CO2H

C6C2N

N

L-Trp

indole.C2N

NH2

HO L-Tyr

CO2H

H2N L-Orn H2N

CO2H L-Lys

N

NH2

NH2

C4N

N

N

C5N

N

Figure 2.2 (Continued)

•

•

•

•

methylerythritol phosphate. Mevalonate itself is the product from three acetyl-CoA molecules, but only five of mevalonate’s six carbon atoms are used, the carboxyl group being lost. The alternative precursor, methylerythritol phosphate, is formed from a straight-chain sugar derivative, deoxyxylulose phosphate, which undergoes a skeletal rearrangement to form the branched-chain isoprene unit. C6 C3 : This refers to a phenylpropyl unit and is obtained from the carbon skeleton of either l-phenylalanine or l-tyrosine, two of the shikimate-derived aromatic amino acids. This, of course, requires loss of the amino group. The C3 side-chain may be saturated or unsaturated, and may be oxygenated. Sometimes the side-chain is cleaved, removing one or two carbon atoms. Thus, C6 C2 and C6 C1 units represent modified shortened forms of the C6 C3 system. C6 C2 N: Again, this building block is formed from either l-phenylalanine or l-tyrosine, with l-tyrosine being by far the more common precursor. In the elaboration of this unit, the carboxyl carbon of the amino acid is removed. indole.C2 N: The third of the aromatic amino acids is l-tryptophan. This indole-containing system can undergo decarboxylation in a similar way to l-phenylalanine and l-tyrosine, so providing the remainder of the skeleton as an indole.C2 N unit. C4 N: The C4 N unit is usually found as a heterocyclic pyrrolidine system and is produced from the non-protein amino acid l-ornithine. In marked contrast to the C6 C2 N and indole.C2 N units described above,

ornithine supplies not its α-amino nitrogen, but the δ-amino nitrogen. The carboxylic acid function and the α-amino nitrogen are both lost. • C5 N: This is produced in exactly the same way as the C4 N unit, but using l-lysine as precursor. The ε-amino nitrogen is retained, and the unit is commonly found as a piperidine ring system. These eight building blocks will form the basis of many of the natural product structures discussed in the following chapters. Simple examples of how compounds can be visualized as a combination of building blocks are shown in Figure 2.3. At this stage, it is inappropriate to justify why a particular combination of units is used, but this aspect should become clear as the pathways are described. A word of warning is also necessary. Some natural products have been produced by processes in which a fundamental rearrangement of the carbon skeleton has occurred. This is especially common with structures derived from isoprene units, and it obviously disguises some of the original building blocks from immediate recognition. The same is true if one or more carbon atoms are removed by oxidation reactions.

THE CONSTRUCTION MECHANISMS Natural product molecules are biosynthesized by a sequence of reactions which, with very few exceptions, are catalysed by enzymes. Enzymes are protein molecules which facilitate chemical modification of substrates by virtue of their specific binding properties conferred by

12 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH

OH

HO Glucose

CO2H

O

OH

O

O

3 × C5

HO2C

O

Me

OMe

MeO

OMe podophyllotoxin 2 × C6 C 3 + 4 × C 1

N

N

MeO CO2H

O O naringin

C6C3 + 3 × C2 + sugars

MeO OH

O O

OH

parthenolide

4 × C2

O

Rhamnose O

orsellinic acid

O

CO2Me

MeO

N Me

O

tetrahydrocannabinolic acid

papaverine

lysergic acid

O cocaine

6 × C2 + 2 × C5

C6C2N + (C6C2) + 4 × C1

indole.C2N + C5 + C1

C4N + 2 × C2 + (C6C1) + 2 × C1

NH

MeO

C6C3

C6C3

Figure 2.3 the particular combination of functional groups in the constituent amino acids. In many cases, a suitable cofactor, e.g. NAD+ , PLP, HSCoA (see below), as well as the substrate, may also be bound to participate in the transformation. Although enzymes catalyse some fairly elaborate and sometimes unexpected changes, it is generally possible to account for the reactions using established chemical principles and mechanisms. As we explore the pathways to a wide variety of natural products, the reactions will generally be discussed in terms of chemical analogies. Enzymes have the power to effect these transformations more efficiently and more rapidly than the chemical analogy, and also under very much milder conditions. Where relevant, they also carry out reactions in a stereospecific manner. Some of the important reactions frequently encountered are now described.

Alkylation Reactions: Nucleophilic Substitution The C1 methyl building unit is supplied from l-methionine and is introduced by a nucleophilic substitution reaction. In nature, the leaving group is enhanced by converting l-methionine into S -adenosylmethionine (SAM, AdoMet) [Figure 2.4(a)]. This gives a positively charged sulfur and facilitates the SN 2-type nucleophilic substitution mechanism [Figure 2.4(b)]. Thus, O-methyl and N-methyl linkages may be obtained using hydroxyl

and amino functions as nucleophiles. Methionine is subsequently regenerated by the methylation of homocysteine, using N 5 -methyl-tetrahydrofolate (see page 144) as methyl donor. The generation of C-methyl linkages requires the participation of nucleophilic carbon. Positions ortho or para to a phenol group, or positions adjacent to one or more carbonyl groups, are thus candidates for C-methylation [Figure 2.4(c)]. A C5 isoprene unit in the form of dimethylallyl diphosphate (DMAPP) may also act as an alkylating agent, since diphosphate is a good leaving group [Figure 2.4(d)]. Although a similar SN 2 displacement may be proposed, the available evidence points to an SN 1 process. DMAPP first ionizes to the resonance-stabilized allylic carbocation, and the nucleophile is able to attack either of the cationic centres. In the majority of cases, the nucleophile attacks the same carbon that loses the diphosphate. C-Alkylation at activated positions using DMAPP is analogous to the C-methylation process above, though by an SN 1 mechanism. Alkylation Reactions: Electrophilic Addition As indicated above, the C5 isoprene unit in the form of DMAPP can be used to alkylate a nucleophile. In the elaboration of terpenoids and steroids, two or more C5 units are joined together and the reactions are rationalized

Secondary Metabolism: The Building Blocks and Construction Mechanisms Alkylation reactions: nucleophilic substitution (a) formation of SAM Ad = adenosine

NH2

HO

O

O

O

P

P

P

N

O O O CH2 O OH OH OH

triphosphate is a good leaving group

N

N

N

OH

S

N SN2 reaction

N

H3C

ATP

H 2N

S CH2

N

O

N

HO

OH

CO2H

CO2H L-Met

NH2

E1

HO H3C

Ad

S-adenosylmethionine (SAM, AdoMet)

NH2

E1: SAM synthetase

(b) O- and N-alkylation using SAM; regeneration of methionine Ad OH or

H3C

NH2

S

CO2H SAM

Ad

SN2 reaction O CH3 H

E1

NH2

–

H+

neutral molecule is good leaving group

S

CO2H

HS

E2 NH2 S-adenosylhomocysteine (SAH)

NH2 homocysteine

O CH3 or

NH

CO2H

E3

N5-methyl-FH4 FH4

CH3 H3C

E1: methyltransferase E2: SAH hydrolase E3: methionine synthase

CO2H

S

NH2 L-Met

(c) C-alkylation using SAM Ad

ortho (and para) positions are activated by OH

H3C

S

CO2H NH2

carbonyl groups increase acidity and allow formation of enolate anion

O

Ad H

H3C

S

CO2H NH2

O

OH

(d) O-alkylation using DMAPP OH diphosphate is good leaving group

SN1 reaction

OH

PPO resonance-stabilized allylic carbocation

dimethylallyl diphosphate (DMAPP) – H+ O

O H

Figure 2.4

HO

– H+

O

13

14 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition in terms of carbocation chemistry, including electrophilic addition of carbocations onto alkenes. DMAPP may ionize to generate a resonance-stabilized allylic carbocation as shown in Figure 2.4(d), and this can then react with an alkene, e.g. isopentenyl diphosphate (IPP), as depicted in Figure 2.5(a). The resultant carbocation may then lose a proton to give the uncharged product geranyl diphosphate (GPP). Where the alkene and carbocation functions reside in the same molecule, this type of mechanism can also be responsible for cyclization reactions [Figure 2.5(a)].

The initial carbocation may be generated by a number of mechanisms, important examples being loss of a leaving group, especially diphosphate (i.e. SN 1-type ionization), protonation of an alkene, and protonation/ring opening of epoxides [Figure 2.5(b)]. SAM may also alkylate alkenes by an electrophilic addition mechanism, adding a C1 unit, and generating an intermediate carbocation; this is simply an extension of the protonation reaction. The final carbocation may be discharged by loss of a proton (giving an alkene or sometimes a cyclopropane ring) or by quenching with a suitable nucleophile, especially water [Figure 2.5(c)].

Alkylation reactions: electrophilic addition (a) inter- and intra-molecular additions electrophilic addition of cation onto alkene: intermolecular addition – H+ OPP isopentenyl diphosphate (IPP)

OPP

OPP

H

geranyl diphosphate (GPP)

intramolecular addition: cyclization

(b) generation of carbocation R R R

L

H

R

R

R

R

R

R

R

R

R

R

R O R

R OH R

R H

R

H loss of leaving group

protonation of alkene

R

R

R

protonation and ring opening of epoxide

Ad H3C

S

CO2H SAM

R

R

R

R

NH2 R

CH3 R

R

R

methylation of alkene via SAM

(c) discharge of carbocation R

H

H

R

R

R

R

R R

R

H2O R

R

H R

loss of proton

Figure 2.5

H

R

R

R

R

OH R H

H R

cyclization / loss of proton

R

R

R

R

R

quenching with nucleophile (water)

Secondary Metabolism: The Building Blocks and Construction Mechanisms

15

Wagner–Meerwein rearrangements

R

H

H

1,2-hydride shift R

H

H R R tertiary carbocation

R R secondary carbocation

H

R

1,2-methyl shift R

R R secondary carbocation 1,3-hydride shift

1,2-alkyl shift

H tertiary carbocation, but strained 4-membered ring

H R R tertiary carbocation

secondary carbocation, but reduced ring strain in 5-membered ring

H

tertiary carbocation

H

H

resonance-stabilized allylic cation

a series of concerted 1,2hydride and methyl shifts

H

H

H

H

Figure 2.6 Wagner–Meerwein Rearrangements A wide range of structures encountered in natural terpenoid and steroid derivatives can only be rationalized as originating from C5 isoprene units if some fundamental rearrangement process has occurred during biosynthesis. These rearrangements have, in many cases, been confirmed experimentally, and are almost always consistent with the participation of carbocation intermediates. Rearrangements in chemical reactions involving carbocation intermediates, e.g. SN 1 and E1 reactions, are not uncommon and typically consist of 1,2-shifts of hydride, methyl, or alkyl groups. Occasionally, 1,3or longer shifts are encountered. These shifts, termed Wagner–Meerwein rearrangements, are readily rationalized in terms of generating a more stable carbocation or relaxing ring strain (Figure 2.6). Thus, tertiary carbocations are favoured over secondary carbocations, and the usual objective in these rearrangements is to achieve tertiary status at the positive centre. However, a tertiary to secondary transition might be favoured if the rearrangement allows a significant release of ring strain. These general concepts are occasionally ignored by nature, but it must be remembered that the reactions are enzyme mediated and that carbocations may not exist as discrete species in the transformations. An interesting feature of some biosynthetic pathways, e.g.

that leading to steroids, is a remarkable series of concerted 1,2-migrations rationalized via carbocation chemistry, but entirely a consequence of the enzyme’s participation (Figure 2.6). Aldol and Claisen Reactions The aldol and Claisen reactions both achieve carbon–carbon bond formation; in typical base-catalysed chemical reactions, this depends upon the generation of a resonance-stabilized enolate anion from a suitable carbonyl system (Figure 2.7). Whether an aldol-type or Claisen-type product is formed depends on the nature of X and its potential as a leaving group in the alkoxide anion intermediate. Thus, chemically, two molecules of acetaldehyde yield aldol, whilst two molecules of ethyl acetate can give ethyl acetoacetate. These processes are vitally important in biochemistry for the elaboration of both secondary and primary metabolites, but the enzyme catalysis obviates the need for strong bases, and probably means the enolate anion has little more than transitory existence. Nevertheless, the reactions do appear to parallel enolate anion chemistry and are frequently responsible for joining together of C2 acetate groups. In most cases, the biological reactions involve coenzyme A esters, e.g. acetyl-CoA (Figure 2.8). This is a

16 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Aldol and Claisen reactions O

O R

O

R

X

R

X

H H

H

resonance-stabilized enolate anion

B O R

O

R X

nucleophilic addition of enolate anion onto carbonyl

O loss of leaving group

O

X R H

Claisen-type product

X R H alkoxide anion

X

O

R

X

O

R

X H

R = H, X = OEt, ethyl acetoacetate

H+

H

OH

R

R = X = H, acetaldehyde R = H, X = OEt, ethyl acetate

X

O

if no suitable leaving group, protonation occurs

X R H

aldol-type product R = X = H, aldol

Figure 2.7 ADP HS

ribose CoA O

O

HS

N H cysteamine (2-mercaptoethylamine)

N H

OH pantothenic acid

pantetheine

O

O

O

P

P

N

O O CH2 O OH OH HO HO O P

SCoA

O acetyl-CoA

+

H

O

O

H3C

OH

O O

ester

Et

H3C

O

H3C

O

Et

resonance decreases acidity of a-hydrogens

O OEt

RS− is a better leaving group than RO−

Figure 2.8

H3C

Nu SCoA

N

O

SCoA

thioesters are more acidic than oxygen esters

Nu H3C

H2C

N

N

O

Coenzyme A HSCoA

H3C

adenine NH2

O S

thioester

CoA

H3C

S

CoA

resonance of this type is less favourable in the sulfur ester

Secondary Metabolism: The Building Blocks and Construction Mechanisms nucleophilic attack onto suitable acyl derivative

nucleophilic attack on thioester; thiolate is good leaving group

O Cys

SH

O

O

R

17

S

L Cys

Cys−SH is part of enzyme L is often SCoA

Cys

Nu

R

SH

R

Nu

acyl group is now bound to enzyme as thioester

Figure 2.8 (Continued)

ATP + HCO3

O HO

O

P

loss of biotin–enzyme as leaving group

O O

O

E1a ADP

nucleophilic attack of enolate acetyl-CoA O anion onto carbonyl (enolate) CoAS CoAS CH2

nucleophilic attack onto mixed anhydride

O HN

O OH H OH mixed anhydride

NH

O N HO H

N E1a

H

CO Enz

S biotin–enzyme

E1b

HO N1-carboxybiotin–enzyme

NH H CO Enz

S E1b

acetyl-CoA carboxylase (3 component proteins) E1a: biotin carboxylase E1b: carboxyltransferase Enz: biotin carboxyl carrier protein

O

O

CoAS

+

biotin–enzyme

OH

malonyl-CoA O CH3

SCoA O

CO2 lost in concerted reaction

SCoA O

O CH3

O SCoA

SCoA

acetoacetyl-CoA

SCoA O

O malonyl-CoA H

CH3

O CO2

O

alternatively, enzyme generates transient enolate anion O

O SCoA

CH2

SCoA

O H

B Enz

Figure 2.9 thioester of acetic acid, and it has significant advantages over oxygen esters, e.g. ethyl acetate, for two main reasons. First, the α-methylene hydrogen atoms are now more acidic, comparable in fact to those in the equivalent ketone, and this increases the likelihood of generating an enolate anion. This is explained in terms of electron delocalization in an ester function (Figure 2.8). This type of delocalization is more prominent in the oxygen ester than in the sulfur ester, due to oxygen’s smaller size and closer proximity of the lone pair for overlap with carbon’s orbitals. Second, the thioester has a much more favourable leaving group than the oxygen

ester. The combined effect is to increase the reactivity for both the aldol- and Claisen-type reactions. Thioester linkages also provide a means of covalently bonding suitable substrates to enzymes, prior to enzymic modification, followed by subsequent release (Figure 2.8). Claisen reactions involving acetyl-CoA are made even more favourable by first converting acetyl-CoA into malonyl-CoA by a carboxylation reaction catalysed by the three-component enzyme acetyl-CoA carboxylase. The reaction requires CO2 , ATP and the coenzyme biotin (Figure 2.9). ATP and CO2 (solubilized as bicarbonate, HCO3 − ) form the mixed anhydride, a reaction also

18 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition catalysed by the enzyme, and this is used to carboxylate the coenzyme which is bound in a biotin–enzyme complex. The carboxylation reaction is effectively a nucleophilic attack of the cyclic urea onto the mixed anhydride. Fixation of CO2 by biotin–enzyme complexes is not unique to acetyl-CoA; another important example occurs in the generation of oxaloacetate from pyruvate in the synthesis of glucose from non-carbohydrate sources (gluconeogenesis). The conversion of acetyl-CoA into malonyl-CoA means the α-hydrogen atoms are now flanked by two carbonyl groups, and have increased acidity. Thus, a more favourable nucleophile is provided for the Claisen reaction. No acylated malonic acid derivatives are produced, and the carboxyl group introduced into malonyl-CoA is simultaneously lost by a decarboxylation reaction during the Claisen condensation (Figure 2.9). An alternative rationalization is that decarboxylation of the malonyl ester is used to generate the transient acetyl enolate anion without any requirement for a strong base. Thus, the product formed from acetyl-CoA as electrophile and malonyl-CoA as nucleophile is acetoacetyl-CoA, exactly the same as in the condensation of two molecules of acetyl-CoA. Accordingly, the role of the carboxylation step is clear-cut: the carboxyl activates the α-carbon to facilitate the Claisen condensation and it is immediately removed on completion of this task. By analogy, the chemical Claisen condensation using the enolate anion from diethyl malonate in Figure 2.10 proceeds much more favourably than that using the enolate anion from ethyl acetate. The same acetoacetic acid product can be formed in the malonate condensation by hydrolysis of the acylated malonate intermediate and decarboxylation of the gem-diacid. Analogous carboxylations of some other thioesters may occur; for example, propionyl-CoA may be converted into methylmalonyl-CoA. O CH3

O OEt

CH2

OEt

EtOH

O

CH3

O

O

H+ OEt

O

CH3

OH

acetoacetic acid Δ – CO2

EtOH

CO2Et enolate anion from diethyl malonate

Figure 2.10

Formation of C–N bonds is frequently achieved by condensation reactions between amines and aldehydes or ketones. A typical nucleophilic addition is followed by elimination of water to give an imine or Schiff base [Figure 2.12(a)]. Of almost equal importance is the reversal of this process, i.e. the hydrolysis of imines to amines and aldehydes/ketones [Figure 2.12(b)]. The imine so produced, or more likely its protonated form the iminium cation, can then act as an electrophile in a Mannich reaction [Figure 2.12(c)]. The nucleophile might be provided by an enolate anion, or in many examples by a suitably activated centre in an aromatic ring system. The Mannich reaction is encountered throughout alkaloid biosynthesis, and in its most general form involves combination of an amine (primary or secondary), an aldehyde or ketone, and a nucleophilic carbon. Secondary amines will react with the carbonyl compound to give an iminium cation (quaternary Schiff base) directly; thus, the additional protonation step is not necessary. It should be appreciated that the Mannich-like addition reaction in Figure 2.12(c) is little different from

CH3

O

NaOEt OEt

OEt

Imine Formation and the Mannich Reaction

ethyl acetoacetate

enolate anion from ethyl acetate O

O

NaOEt

Both the reverse aldol and reverse Claisen reactions may be encountered in the modification of natural product molecules. Such reactions remove fragments from the basic skeleton already generated, but may extend the diversity of structures. The reverse Claisen reaction is a prominent feature of the β-oxidation sequence for the catabolic degradation of fatty acids (Figure 2.11): a C2 unit as acetyl-CoA is cleaved off from a fatty acid chain, leaving it two carbon atoms shorter in length. Though the terminology β-oxidation strictly refers to the introduction of the new carbonyl group, it is usually understood to include the chain shortening.

O

CH3

O

H+ OEt

CO2Et acylated diethyl malonate

O

CH3 ester hydrolysis

O HO

O

gem-diacid

H

thermal decarboxylation

Secondary Metabolism: The Building Blocks and Construction Mechanisms β-Oxidation of fatty acids dehydrogenation; hydrogen atoms passed to FAD

stereospecific hydration of double bond

FAD FADH2

O

H2O

O

HO H

E R

SCoA

R

E1

SCoA

dehydrogenation; hydrogen atoms passed to NAD+

R S

E2

NAD+ NADH

O

SCoA

O

R

E3

fatty acyl-CoA (chain length C2n) E1: E2: E3: E4:

O

SCoA HSCoA

reverse Claisen E4 reaction O

acyl-CoA dehydrogenase enoyl-CoA hydratase β-hydroxyacyl-CoA dehydrogenase thiolase

R

O SCoA

CH3

SCoA

acetyl-CoA

fatty acyl-CoA (chain length C2n–2)

Figure 2.11 (a) Imine formation equilibrium between protonated species; proton may be on N or O

nucleophilic attack onto carbonyl H O H

R NH2

R

– H+, + H+ N

H

primary amine

O H

NH

R1

R1 secondary amine

N

OH

– H+, + H+ N

H

H

R1

H

R NH2

H

H 2O

HO

OH

(c) Mannich reaction nucleophilic addition onto iminium ion R

R N

H iminium cation

Figure 2.12

N

C H carbanion-type nucleophile, e.g. enolate anion

OH2

N R1 iminium cation (quaternary Schiff base)

loss of amine leaving group; formation of carbonyl

R N

R N

H

R N

imine (Schiff base)

R

OH

(b) Imine hydrolysis nucleophilic attack onto imine or protonated imine

R N

OH2

H

H

R

R

elimination of water

R

19

20 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition H R

H+

N

R

R

imine–enamine tautomerism

N

N H

H imine

enamine R N

R N H

H

R

protonated imine

H

NHR N

enamine

aldol-type reaction between two imine systems behaving as enamine–iminium ion pair

aldol-type addition product

Figure 2.13

reductive amination

NH3 H2O NAD(P)H NAD(P)+

HO2C

this involves formation and reduction of an intermediate imine

HO2C

O

H NH2

O

NH3

NAD(P)H

NH

NH2

E1

HO2C 2-oxoglutaric acid

HO2C glutamic acid L-Glu

E1: glutamate dehydrogenase

Figure 2.14 nucleophilic addition to a carbonyl group. Indeed, the imine/iminium cation is merely acting as the nitrogen analogue of a carbonyl/protonated carbonyl. To take this analogy further, protons on carbon adjacent to an imine group will be acidic, as are those α to a carbonyl group, and the isomerization of an imine to the enamine shown in Figure 2.13 is analogous to keto–enol tautomerism. Just as two carbonyl compounds can react via an aldol reaction, so can two imine systems; and this is indicated in Figure 2.13. Often, aldehyde/ketone substrates in enzymic reactions become covalently bonded to an amino group in the enzyme through imine linkages; in so doing they lose none of the carbonyl activation as a consequence of the new form of bonding. Amino Acids and Transamination The synthesis of amino acids depends upon the amination of the Krebs cycle intermediate 2-oxoglutaric acid to glutamic acid, a process of reductive amination (Figure 2.14). The reaction involves imine formation and subsequent reduction. The reverse reaction is also important in amino acid catabolism. Glutamic acid can then participate in the formation of other amino acids via transamination, the exchange of

the amino group from an amino acid to a keto acid. This provides the most common process for the introduction of nitrogen into amino acids, and for the removal of nitrogen from them. The reaction is catalysed by a transaminase enzyme using the coenzyme pyridoxal phosphate (PLP) and features an imine intermediate (aldimine) with the aldehyde group of PLP (Figure 2.15). Protonation of the pyridine nitrogen (as would occur at physiological pH) provides an electron sink and makes the α-hydrogen of the original amino acid now considerably more acidic. This can be removed, generating a dihydropyridine ring system. Reprotonation then produces a new imine (ketimine) and also restores aromaticity in the pyridine ring. However, because of the conjugation, it allows protonation at a position that is different from where the proton was originally lost. The net result is that the imine double bond has effectively moved to a position adjacent to its original position. Hydrolysis of the new imine function generates a keto acid and pyridoxamine phosphate. The remainder of the sequence is now a reversal of this process. This can now transfer the amine function from pyridoxamine phosphate to another keto acid. The glutamic acid–2-oxoglutaric acid couple provides the usual donor–acceptor molecules for the amino group, and thus glutamate transaminase is the most important

Secondary Metabolism: The Building Blocks and Construction Mechanisms

21

Transamination HO2C

H NH2

HO2C

HO2C

PLP

O

O R

HO2C

E1

H NH2

R

HO2C

HO2C

glutamic acid

keto acid

R

2-oxoglutaric acid

amino acid

E1: glutamate transaminase

CO2H

R

H NH2

CO2H O NH2

CHO OH

PO

OH

PO N

CH3

N CH3 pyridoxamine P

pyridoxal P (PLP)

hydrolysis of imine to keto acid and amine

formation of imine from aldehyde and amino acid R CO2H protonation of ring H nitrogen enhances N acidity of α-hydrogen

R

N

N

O

CH3

CO2H

H2N H CO2H

E1

HO2C CO2H glutamic acid O HO2C

N OH N

CH3

ketimine

O

H2N H

R

CO2H

PO

H

H aldimine

R

OH

PO

CH3

R protonation restores aromaticity

N

H

OH

PO

CO2H

R′

H2N CO2H

E2

amino group transferred

H2N H CO2H

2-oxoglutaric acid

R′

CO2H

E1: glutamate transaminase E2: a transaminase

Figure 2.15 of the transaminases. Transamination allows the amino group to be transferred from glutamic acid to a suitable keto acid, or from an amino acid to 2-oxoglutaric acid in the reverse mode. It is thus possible to transfer the amino group of one amino acid, which may be readily

available from the diet, to provide another amino acid, which may be in short supply (Figure 2.15). For many organisms, this means it is no longer necessary to be able to carry out the ammonia-dependent reductive amination of 2-oxoglutaric acid.

22 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition transamination sequence of Figure 2.15. Decarboxylation of the intermediate aldimine is facilitated in the same way as loss of the α-hydrogen in the transamination sequence. The protonated nitrogen acts as an electron sink and the conjugated system allows loss of the carboxyl proton with subsequent bond breaking and loss of CO2 . After protonation of the original α-carbon, the amine (decarboxylated amino acid) is released from the coenzyme by hydrolysis of the imine function; PLP is regenerated. β-Keto acids are thermally labile and rapidly decarboxylated in vitro via a cyclic mechanism which proceeds through the enol form of the final ketone [Figure 2.16(b)]. Similar reactions are found in nature, though whether cyclic processes are necessary is not clear. ortho-Phenolic acids also decarboxylate readily in vitro and in vivo, and it is again possible to invoke

Decarboxylation Reactions Many pathways to natural products involve steps which remove portions of the carbon skeleton. Although two or more carbon atoms may be cleaved off via the reverse aldol or reverse Claisen reactions mentioned above, by far the most common degradative modification is loss of one carbon atom by a decarboxylation reaction. Decarboxylation is a particular feature of the biosynthetic utilization of amino acids, and it has already been indicated that several of the basic building blocks (e.g. C6 C2 N and indole.C2 N) are derived from an amino acid via loss of the carboxyl group. This decarboxylation of α-amino acids is also a PLP-dependent reaction (compare transamination) and is represented as in Figure 2.16(a). This similarly depends on imine formation and shares features of the

Decarboxylation reactions (a) α-amino acids O R

CO2H

PLP

H

H NH2 imine formation

R N

R

H

O

N

protonation restores aromaticity

H N

decarboxylation OH

PO

H

OH

PO N

CH3

H

R

OH N

CH3

H

H

(b) β-keto acids 6-membered hydrogen-bonded system

O

.... H

R

O β-keto acid

CO2

O keto tautomer ≡ β-keto acid H

O

HO O H

O keto tautomer

Figure 2.16

CO2

OH

H

phenolic acid (i.e. enol tautomer)

CO2H

CH2

intermediate enol

O

CO2H

HO

R

O .... H

OH

keto–enol tautomerism O

OH

O

H

CO2

R

PLP

R NH2

N

PO

CH3

H

CH3

hydrolysis of imine to aldehyde and amine

Secondary Metabolism: The Building Blocks and Construction Mechanisms

23

(c) α-keto acids pyruvic acid → acetaldehyde (pyruvate decarboxylase)

nucleophilic attack of carbanion onto carbonyl: aldol-type reaction

acidic hydrogen B

H 3C

H

NH2 N

N

O

S

N

N

decarboxylation of b-iminium acid

H

O

HO H 3C

CO2H S

O

N

H 3C

H

OH H

N

S

S

OPP TPP ylid (TPP anion)

thiamine diphosphate (TPP)

enamine enamine–imine tautomerism O

TPP ylid regenerated

pyruvic acid → acetyl-CoA (pyruvate dehydrogenase) H H3C

OH S

enamine

H H3C

O

H

N

S

H3C

O

N

H H S

iminium cation O

S S

N

H3C

reverse aldol-type reaction

OH

O S

enzyme-bound lipoic acid

S

enamine attacks S of lipoic acid fragment with S–S bond fission

enzyme-bound lipoic acid

acetyl group displaced by coenzyme A O HS

HS H3C

FAD

HSCoA

O

S

OH

S

H 3C

SCoA HS

acetyl-CoA HS

N

S

N

S

reverse aldol-type reaction regenerates ylid and leaves acetyl group attached to dihydrolipoic acid

original lipoic acid fragment has become reduced to dithiol; oxidation regenerates the enzyme-bound lipoic acid

Figure 2.16 (Continued) a cyclic β-keto acid tautomer of the substrate. The corresponding decarboxylation of para-phenolic acids cannot have a cyclic transition state, but the carbonyl group in the proposed keto tautomer activates the system for decarboxylation. The acetate pathway frequently yields structures containing phenol and carboxylic acid

functions; thus, decarboxylation reactions may feature as further modifications. Although the carboxyl group may originate by hydrolysis of the thioester portion of the acetyl-CoA precursor, there are also occasions when a methyl group can be sequentially oxidized to a carboxyl, which then subsequently suffers decarboxylation.

24 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Decarboxylation of α-keto acids is a feature of primary metabolism, e.g. pyruvic acid → acetaldehyde in glycolysis, and pyruvic acid → acetyl-CoA, an example of overall oxidative decarboxylation prior to entry of acetyl-CoA into the Krebs cycle. Both types of reaction depend upon thiamine diphosphate (TPP). TPP is a coenzyme containing a thiazole ring in the form of a thiazolium salt. The proton in the thiazolium ring is relatively acidic and can be removed to generate a carbanion (strictly an ylid, a species with positive and negative charges on adjacent atoms). The ylid can act as a nucleophile and is also a reasonable leaving group. Addition to the carbonyl group of pyruvic acid is followed by decarboxylation, as depicted in Figure 2.16(c), with the positive nitrogen acting as an electron sink. The resulting molecule is an enamine. Tautomerism to the iminium cation is followed by a reverse aldol reaction, which also regenerates the ylid. In the oxidation step of oxidative decarboxylation, the enzyme-bound disulfide-containing coenzyme lipoic acid is also involved. The intermediate enamine in Figure 2.16(c), instead of accepting a proton, is used to attack a sulfur in the lipoic acid moiety with subsequent S–S bond fission, thereby effectively reducing the lipoic acid fragment. This allows regeneration of the TPP ylid by a reverse aldol reaction, leaving the acetyl group bound to the dihydrolipoic acid. This acetyl group is then released as acetyl-CoA by displacement with the thiol coenzyme A. The bound dihydrolipoic acid fragment is then reoxidized to restore its function. An exactly equivalent reaction is encountered in the Krebs cycle in the conversion of 2-oxoglutaric acid into succinyl-CoA.

then a pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+ ) or nicotinamide adenine dinucleotide phosphate (NADP+ ), tends to be utilized as hydrogen acceptor. One hydrogen from the substrate (that bonded to carbon) is transferred as hydride to the coenzyme and the other (as a proton) is passed to the medium (Figure 2.17). NAD(P)+ may also be used in the oxidations:

Oxidation and Reduction Reactions

the coenzyme used as acceptor is usually a flavin nucleotide, flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). These entities are bound to the enzyme in the form of a flavoprotein and take up two hydrogen atoms, represented in Figure 2.18 as being derived by addition of hydride from the substrate and a proton from the medium. Alternative mechanisms have also been proposed, however. Reductive sequences involving flavoproteins may be represented as the reverse reaction in Figure 2.18. NADPH may also be employed as a coenzyme in the reduction of a carbon–carbon double bond. These oxidation reactions employing pyridine nucleotides and flavoproteins are especially important in primary metabolism in liberating energy from fuel molecules in the form of ATP. The reduced coenzymes formed in the process are normally reoxidized via the electron transport chain of oxidative phosphorylation, so that the hydrogen atoms eventually pass to oxygen, giving water.

Changes to the oxidation state of a molecule are frequently carried out as a secondary metabolite is synthesized or modified. The processes are often complex and not always completely understood, but the following general features are recognized. The processes may be classified according to the type of enzyme involved and their mechanism of action. Dehydrogenases Dehydrogenases remove two hydrogen atoms from the substrate, passing them to a suitable coenzyme acceptor. The coenzyme system involved can generally be related to the functional group being oxidized in the substrate. Thus if the oxidation process is CH

OH

C O

H C O

CH NH2

CO2H

C

NH

The reverse reaction, i.e. reduction, is also indicated in Figure 2.17, and may be compared with the chemical reduction process using complex metal hydrides, e.g. LiAlH4 or NaBH4 , namely nucleophilic addition of hydride and subsequent protonation. The reduced forms NADH and NADPH are conveniently regarded as hydride-donating reducing agents. During the reduction sequence, there is stereospecific transfer of hydride from a prochiral centre on the dihydropyridine ring, and it is also delivered to the carbonyl compound in a stereospecific manner. In practice, NADPH is generally employed in reductive processes, whilst NAD+ is used in oxidations. Should the oxidative process be the conversion CH2

CH2

CH

CH

Secondary Metabolism: The Building Blocks and Construction Mechanisms

25

Dehydrogenases: NAD+ and NADP+ NH2 N

N N

O

N RO

CH2 O

O

CONH2

O

adenine

P

P O O CH2 O OH OH

OH

N

HO

R = H, NAD+ R = P, NADP+

≡

ribose

ribose

P

P

P

OH

O

H

abstraction of hydride from substrate

nicotinamide

H

H

O H H

CONH2

CONH2

dehydrogenase

N

donation of hydride to substrate

N

R NAD+ NADP+

R NADH NADPH

Figure 2.17

Dehydrogenases: FAD and FMN O N

H3C

NH

NH2 N

N N

N HO

O

O CH2 O O

H3C

P O O OH OH

OH

N

N

P

OH

O

adenine

≡ OH

ribose

flavin P

ribitol

P

OH

FMN FAD H

H

abstraction of hydride from substrate H H3C

N

H3C

N R FAD FMN

Figure 2.18

H

O H3C

NH N H

donation of hydride to substrate

O

dehydrogenase

H3C

O

N

NH

N

N

R

H

FADH2 FMNH2

O

26 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Monooxygenases

Oxidases OH

O +

½ O2

OH ortho-quinol

+

H

H2O

O ortho-quinone H

O

OH + OH para-quinol

½ O2

+

O2

O2

OH

NADPH

H2O O2 NADPH

O para-quinone

OH

NADPH

O

aliphatic hydroxylation

aromatic hydroxylation

epoxidation of alkene

Figure 2.20 Figure 2.19 Oxidases Oxidases also remove hydrogen from a substrate, but pass these atoms to molecular oxygen or to hydrogen peroxide, in both cases forming water. Oxidases using hydrogen peroxide are termed peroxidases. Mechanisms of action vary and need not be considered here. Important transformations in secondary metabolism include the oxidation of ortho- and para-quinols to quinones (Figure 2.19), and the peroxidase-induced phenolic oxidative coupling processes (see page 28). Monooxygenases Oxygenases catalyse the direct addition of oxygen from molecular oxygen to the substrate. They are subdivided into mono- and di-oxygenases according to whether just one or both of the oxygen atoms are introduced into the substrate. With monooxygenases, the second oxygen atom from O2 is reduced to water by an appropriate hydrogen donor, e.g. NADH, NADPH, or ascorbic acid (vitamin C). In this respect they may also be considered to behave as oxidases, and the term ‘mixed-function oxidase’ is also used for these enzymes. Especially important examples of these enzymes are the cytochrome P-450-dependent monooxygenases. These are frequently involved in biological hydroxylations, either in biosynthesis or in the mammalian detoxification and metabolism of foreign compounds such as drugs, and such enzymes are thus termed hydroxylases. Cytochrome P-450 is named after its intense absorption band at 450 nm when exposed to CO, which is a powerful inhibitor of these enzymes. It contains an iron–porphyrin complex (haem) which is bound to the enzyme. A redox change involving the Fe atom allows binding and the cleavage of molecular oxygen to oxygen atoms, with subsequent transfer of one

atom to the substrate. Many such systems have been identified, capable of hydroxylating aliphatic or aromatic systems, as well as producing epoxides from alkenes (Figure 2.20). In most cases, NADPH features as hydrogen donor. Aromatic hydroxylation catalysed by monooxygenases (including cytochrome P-450 systems) probably involves arene oxide (epoxide) intermediates (Figure 2.21). An interesting consequence of this mechanism is that, when the epoxide opens up, the hydrogen atom originally attached to the position which becomes hydroxylated can migrate to the adjacent carbon on the ring. A high proportion of these hydrogen atoms are subsequently retained in the product, even though enolization allows some loss of this hydrogen. This migration is known as the NIH shift, having been originally observed at the US National Institutes of Health. The oxidative cyclization of an ortho-hydroxymethoxysubstituted aromatic system giving a methylenedioxy group is also known to involve a cytochrome P-450-dependent monooxygenase. This enzyme hydroxylates the methoxy methyl to yield an intermediate that is a hemiacetal of formaldehyde; this can cyclize to the methylenedioxy bridge (the acetal of formaldehyde) by an ionic mechanism (Figure 2.22). Dioxygenases Dioxygenases introduce both atoms from molecular oxygen into the substrate, and are frequently involved in the cleavage of carbon–carbon bonds, including aromatic rings. Cyclic peroxides (dioxetanes) have been proposed as intermediates (Figure 2.23), and though evidence points against dioxetanes, they do provide a simple appreciation of the reaction outcome. Oxidative cleavage of aromatic rings typically employs catechol (1,2-dihydroxy) or

Secondary Metabolism: The Building Blocks and Construction Mechanisms

27

NIH shift R

hydride migration NIH shift

R

R

R

H H H

H

O

arene oxide

H

H

H O

– H+

R loss of labelled hydrogen

O

H – H+

enolization R

H

H

OH

retention of labelled hydrogen

OH

Figure 2.21

Methylenedioxy groups H O

O2 CH3 NADPH

OH ortho-hydroxymethoxy derivative

O

CH2

OH hemiacetal of formaldehyde

OH

nucleophilic attack onto carbonyl equivalent O

CH2

O H

O O methylenedioxy derivative

Figure 2.22 quinol (1,4-dihydroxy) substrates, and in the case of catechols, cleavage may be between or adjacent to the two hydroxyls, giving products containing aldehyde and/or carboxylic acid functionalities (Figure 2.23). Some dioxygenases utilize two acceptor substrates and incorporate one oxygen atom into each. Thus, 2-oxoglutarate-dependent dioxygenases hydroxylate one substrate, whilst also transforming 2-oxoglutarate into succinate with the release of CO2 (Figure 2.24). 2-Oxoglutarate-dependent dioxygenases also require as cofactors Fe2+ to generate an enzyme-bound iron–oxygen complex, and ascorbic acid (vitamin C) to reduce this complex subsequently. Amine Oxidases In addition to the oxidizing enzymes outlined above, those which transform an amine into an aldehyde, the amine oxidases, are frequently involved in metabolic pathways. These include monoamine oxidases and diamine oxidases. Monoamine oxidases utilize a flavin nucleotide, typically FAD, and molecular oxygen; the transformation

involves initial dehydrogenation to an imine, followed by hydrolysis to the aldehyde and ammonia (Figure 2.25). Diamine oxidases require a diamine substrate and oxidize at one amino group using molecular oxygen to give the corresponding aldehyde. Hydrogen peroxide and ammonia are the other products formed. The aminoaldehyde so formed then has the potential to be transformed into a cyclic imine. Baeyer–Villiger Monooxygenases The chemical oxidation of ketones by peracids, the Baeyer–Villiger oxidation, yields an ester, and the process is known to involve migration of an alkyl group from the ketone (Figure 2.26). For comparable ketone → ester conversions known to occur in biochemistry, FAD-dependent monooxygenase enzymes requiring NADPH and O2 appear to be involved. This leads to formation of a flavin–peroxide and a mechanism similar to that for the chemical Baeyer–Villiger oxidation may thus operate. The oxygen atom introduced thus originates from O2 .

28 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Dioxygenases

+

O

O2

O

O O

dioxetane O2

OH

O

CHO

O

CHO

O

CO2H

O

CO2H

O

CHO

O

CO2H

OH

O2

OH

cleavage between hydroxyls: intradiol dioxygenase

OH O2

OH

OH OH

OH

cleavage adjacent to hydroxyls: extradiol dioxygenase

OH

Figure 2.23

2-Oxoglutarate-dependent dioxygenases HO2C

O2

O

CO2

CO2H

R H

HO2C 2-oxoglutaric acid

R OH

2-oxoglutaratedependent HO2C dioxygenase succinic acid

Figure 2.24 Amine oxidases FAD, O2 NH3 RCH2NH2

monoamine oxidase O2, H2O

H2N

n

RCHO

NH3, H2O2

NH2 diamine oxidase

H2N

CHO n

Figure 2.25

be brought about by oxidase enzymes, including peroxidase and laccase systems, known to be radical generators. Other enzymes catalysing phenolic oxidative coupling have been characterized as cytochrome P-450-dependent proteins, requiring NADPH and O2 cofactors, but no oxygen is incorporated into the substrate. Hydrogen abstraction from a phenol (a one-electron oxidation) gives the radical, and the unpaired electron can then be delocalized via resonance forms in which the free electron is dispersed to positions ortho and para to the original oxygen function (Figure 2.27). These phenol-derived radicals do not propagate a radical chain reaction, but instead are quenched by coupling with other radicals. Coupling of two of these resonance structures in various combinations gives a range of dimeric systems, as exemplified in Figure 2.27. The final products indicated are then derived by enolization, which restores aromaticity to the rings. Thus, carbon—carbon bonds involving positions ortho or para to the original phenols, or ether linkages, may be formed. The reactive dienone systems formed as intermediates may in some cases be attacked by other nucleophilic groupings, extending the range of structures ultimately derived from this basic reaction sequence.

Phenolic Oxidative Coupling Many natural products are produced by the coupling of two or more phenolic systems, in a process readily rationalized by means of radical reactions. The reactions can

Halogenation Reactions Halogenated natural products are widespread, though not particularly common; only a handful of examples are

Secondary Metabolism: The Building Blocks and Construction Mechanisms

Baeyer–Villiger monooxygenases

carbonyl reforms: alkyl group migrates from carbon to adjacent oxygen

nucleophilic attack of peracid onto carbonyl H

Baeyer–Villiger oxidation

O

O

R1

R2

O O peracid

ketone

O

R1

O2, NADPH R2

H

HO O

O O HO

O HO

(HOO–flavin)

R1

R2

R1

O

29

R1

O ester

O

R2

O

O flavin R1

R2

O

R2

Figure 2.26

Phenolic oxidative coupling OH

O

O

O –H

resonance-stabilized radical

phenol

O O

O

O

H

H

H

O

O

H

H H

O

bis-dienone

coupling of two radicals

×2

×2

H O

bis-dienone

keto tautomers

bis-dienone enolization

OH

OH

OH

OH

OH

O enol tautomers

OH ether linkage

ortho–ortho coupling

HO ortho–para coupling

para–para coupling

Figure 2.27 found in this book. The nature of the halogen depends very much upon the relative amount of halide available in the environment, and marine organisms are more likely to synthesize brominated compounds than terrestrial organisms, which tend to produce chlorinated derivatives. Fluorine and iodine are also encountered. Despite halogenation being relatively uncommon, a number of different enzymic mechanisms have been identified for

the process. It is not considered appropriate to present detailed descriptions of these processes, but merely to indicate their general nature. Hypohalite (HOX) is the halogenating species produced by haloperoxidases and flavin-dependent halogenases. This may be considered as a source of electron-deficient halogen (X+ ) that can react with an electron-rich centre in the substrate. Free HOX is not

30 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Glycosylation reactions (a) O-glucosylation HO HO UTP

OH O

HO HO

H HO

ROH

OH O

H HO

O

E1

SN2 reaction

P

P

O

O

OH

glucose 1-P

HO HO

Enz

OH O

B H

HO

CH2 O O HO

E1: UTP-glucose 1-phosphate uridylyltransferase E2: glucosyltransferase

SN2

ribose

HO HO

N

E2

UDP

OR HO

H O-β-D-glucoside

uracil OH

uridine uridine diphosphoglucose UDPglucose OH O

HO HO

OH O

HN

O

O OH

OP

O

Enz B

HO

OPPU

ROH

HO HO

SN2

double SN2 process leads to retention of configuration

OH O

H HO

H

OR

O-α-D-glucoside

ROH HOPPU = uridine diphosphate

(b) C-glucosylation

HO HO

HO OH O H HO

HO HO

OH

OH HO O HO

H OH C-β-D-glucoside

OPPU UDPglucose

(c) hydrolysis of O-glucosides HO HO

OH O

H+ OR

HO

H O-β-D-glucoside

HO HO

OH O

H O

HO

H

R

HO HO

OH O b HO

a

OH2

b

HO HO

OH H O OH HO

H

H β-D-glucose

OH2 a

HO HO

OH O H HO

OH α-D-glucose

HO HO

OH OH OH HO

H

Figure 2.28 usually released; this would not allow regiospecific halogenation. The two types of enzyme require other cofactors, H2 O2 in the case of haloperoxidases or reduced FAD and molecular oxygen for the flavin-dependent halogenases. Halogen radical (X· ) appears to be the

active species involved in halogenation catalysed by 2-oxoglutarate-dependent halogenases. Sometimes, halide anion (X− ) can be employed as the halogenating species, acting as a nucleophile at a suitable electrophilic centre.

Secondary Metabolism: The Building Blocks and Construction Mechanisms Glycosylation Reactions The widespread occurrence of glycosides and polysaccharides requires processes for attaching sugar units to a suitable atom of an aglycone to give a glycoside, or to another sugar giving a polysaccharide. Linkages tend to be through oxygen, although they are not restricted to oxygen, since S-, N-, and C-glycosides are well known. The agent for glycosylation is a nucleoside diphosphosugar, e.g. uridine diphosphoglucose (UDPglucose). Uridine is the nucleoside most frequently employed, though others are utilized depending upon organism and metabolite. A wide variety of sugars, including uronic acids (see page 488), may be transferred via this general mechanism. UDPglucose is synthesized from glucose 1-phosphate and uridine triphosphate (UTP), and the glucosylation process can be envisaged as a simple nucleophilic displacement reaction of SN 2 type [Figure 2.28(a)]. Since UDPglucose has its leaving group in the α-configuration, the product has the β-configuration, as is most commonly found in natural glucosides. Note, however, that many important carbohydrates, e.g. sucrose and starch, possess

31

α-linkages, and these appear to originate via a double SN 2 process [Figure 2.28(a)]. The N-, S-, and C-glycosides are produced in a similar manner with the appropriate nucleophile. In the case of C-glycosides, a suitable nucleophilic carbon is required, e.g. aromatic systems activated by phenol groups [Figure 2.28(b)], as noted in the C-alkylation process described earlier. The hydrolysis of glycosides is achieved by specific hydrolytic enzymes, e.g. β-glucosidase for β-glucosides and β-galactosidase for β-galactosides. These enzymes mimic the readily achieved acid-catalysed processes [Figure 2.28(c)] and may retain or invert the configuration at the anomeric centre. Under acidic conditions, the product would be an equilibrium mixture of the α- and β-anomeric hemiacetal forms, which can also interconvert via the open-chain sugar. Of particular importance is to note that although O-, N-, and S-glycosides may be hydrolysed by acid, C-glycosides are stable to acid. C-Glycosylation introduces a new carbon–carbon linkage, and cleavage requires oxidation, not hydrolysis.

Box 2.1 Some Vitamins Associated with the Construction Mechanisms Vitamin B1 Vitamin B1 (thiamine) (Figure 2.29) is a water-soluble vitamin with a pyrimidinylmethyl-thiazolium structure. It is widely available in the diet, with cereals, beans, nuts, eggs, yeast, and vegetables providing sources. Wheat germ and yeast have very high levels. Dietary deficiency leads to beriberi, characterized by neurological disorders, loss of appetite, fatigue, and muscular weakness. As thiamine diphosphate, vitamin B1 is a coenzyme for pyruvate dehydrogenase, which catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA (see page 23), and for 2-oxoglutarate dehydrogenase, which catalyses a similar reaction on 2-oxoglutarate in the Krebs cycle. It is also a cofactor for transketolase, which transfers a two-carbon fragment between carbohydrates in the pentose phosphate pathway (see page 486). Accordingly, this is a very important component in carbohydrate metabolism. Thiamine is produced synthetically, and foods such as cereals are often enriched. The vitamin is stable in acid solution, but decomposes above pH 5 and is also partially decomposed during normal cooking.

Vitamin B2 Vitamin B2 (riboflavin) (Figure 2.29) is a water-soluble vitamin having an isoalloxazine ring linked to d-ribitol. It is widely available in foods, including liver, kidney, dairy products, eggs, meat, and fresh vegetables. Yeast is a particularly rich source. It is stable in acid solution, not decomposed during cooking, but is sensitive to light. Riboflavin may be produced synthetically, or by fermentation using the yeast-like fungi Eremothecium ashbyii and Ashbya gossypii. Dietary deficiency is uncommon, but manifests itself by skin problems and eye disturbances. Riboflavin is a component of FMN and FAD, coenzymes which play a major role in oxidation–reduction reactions (see page 25). Many key enzymes containing riboflavin (flavoproteins) are involved in metabolic pathways. Since riboflavin contains ribitol and not ribose in its structure, FAD and FMN are not strictly nucleotides, though this nomenclature is commonly accepted and used.

Vitamin B3 Vitamin B3 (nicotinic acid, niacin; Figure 2.29) is a stable, water-soluble vitamin widely distributed in foodstuffs, especially meat, liver, fish, wheat germ, and yeast. However, in some foods, e.g. maize, it may be present in a bound form and is not readily

32 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 2.1 (continued) NH2 N

N

riboflavin (vitamin B2)

OH HO

OH N pyridoxal (vitamin B6)

N OH

N

CONH2 N

nicotinic acid (niacin/vitamin B3)

O

nicotinamide

OH OH

O

OH

HO2C O

NH2

CHO OH

N pyridoxine (pyridoxol)

N

OH

thiamine (vitamin B1)

5′

NH

S

N

HO

CO2H

O N

HO

OH N pyridoxamine

HN

NH

H

N H

OH

OH pantothenic acid (vitamin B5)

H CO2H

S biotin (vitamin H)

Figure 2.29 available. Accordingly, diets based principally on maize may lead to deficiencies. The amino acid tryptophan can be converted in the body into nicotinic acid (see Figure 6.32, page 332) and may provide a large proportion of the requirements. Nicotinic acid is converted into nicotinamide (Figure 2.29), though this compound also occurs naturally in many foods. The term vitamin B3 is often used for the combined nicotinamide–nicotinic acid complement. In the form of the coenzymes NAD+ and NADP+ , nicotinamide plays a vital role in oxidation–reduction reactions (see page 000) and is the most important electron carrier in primary metabolism. Deficiency in nicotinamide leads to pellagra, which manifests itself in diarrhoea, dermatitis, and dementia. Oral lesions and a red tongue may be more noticeable than the other symptoms. Nicotinamide is usually preferred over nicotinic acid for dietary supplements, since there is less risk of vasodilatation. Both are produced synthetically. It is common practice to enrich many foods, including bread, flour, corn, and rice products. Nicotinic acid in large doses can lower both cholesterol and triglyceride concentrations by inhibiting their synthesis. It may be prescribed as an adjunct to statin treatment (see page 98).

Vitamin B5 Vitamin B5 (pantothenic acid; Figure 2.29) is a very widely distributed water-soluble vitamin, though yeast, liver, and cereals provide rich sources. Even though animals must obtain the vitamin through the diet, pantothenic acid deficiency is rare, since most foods provide adequate quantities. Its importance in metabolism is as part of the structure of coenzyme A (see page 16), the carrier molecule essential for carbohydrate, fat, and protein metabolism. Pantothenic acid is specifically implicated in enzymes responsible for the biosynthesis of fatty acids (see page 40), polyketides (see page 70) and some peptides (see page 438).

Vitamin B6 Vitamin B6 covers the three pyridine derivatives pyridoxine (pyridoxol), pyridoxal, and pyridoxamine, and also their 5 -phosphates (Figure 2.29). These are water-soluble vitamins, pyridoxine predominating in plant materials, whilst pyridoxal and pyridoxamine are the main forms in animal tissues. Meat, salmon, nuts, potatoes, bananas, and cereals are good sources. A high proportion of the vitamin activity can be lost during cooking, but a normal diet provides an adequate supply. Vitamin B6 deficiency is usually the result of malabsorption, or may be induced by some drug treatments where the drug may act as an antagonist or increase renal excretion of the vitamin as a side-effect. Symptoms of deficiency are similar to those of niacin (vitamin B3 ) and riboflavin (vitamin B2 ) deficiencies, including eye, mouth, and nose lesions and neurological changes. Synthetic pyridoxine is used for

Secondary Metabolism: The Building Blocks and Construction Mechanisms

33

Box 2.1 (continued) supplementation. Pyridoxal 5 -phosphate is a coenzyme for a large number of enzymes, particularly those involved in amino acid metabolism, e.g. in transamination (see page 20) and decarboxylation (see page 22). It thus plays a central role in the production of the neurotransmitters serotonin and noradrenaline (see page 335), and also of histamine (see page 398). The production of the neurotransmitter γ-aminobutyric acid (GABA) from glutamic acid is also an important pyridoxal-dependent reaction.

Vitamin B12 Vitamin B12 (cobalamins; Figure 2.30) are extremely complex structures based on a corrin ring, which, although similar to the porphyrin ring found in haem, chlorophyll, and cytochromes, has two of the pyrrole rings directly bonded. The central metal atom is cobalt; haem and cytochromes have iron, whilst chlorophyll has magnesium. Four of the six coordinations are provided by the corrin ring nitrogen atoms, and a fifth by a dimethylbenzimidazole moiety. The sixth is variable, being cyano in cyanocobalamin (vitamin B12 ), hydroxyl in hydroxocobalamin (vitamin B12a ), or other anions may feature. Cyanocobalamin is actually an artefact formed as a result of the use of cyanide in purification procedures. The physiologically active coenzyme form of the vitamin is 5 -deoxyadenosylcobalamin (coenzyme B12 ). Vitamin B12 appears to be entirely of microbial origin, with intestinal flora contributing towards human dietary needs. The vitamin is then stored in the liver; animal liver extract has been a traditional source. Commercial supplies are currently obtained by semi-synthesis from the total cobalamin extract of Streptomyces griseus, Propionibacterium species, or other bacterial cultures. This material can be converted into cyanocobalamin or hydroxocobalamin. The cobalamins are stable when protected against light. Foods with a high vitamin B12 content include liver, kidney, meat, and seafood. Vegetables are a poor dietary source, and strict vegetarians may, therefore, risk deficiencies. Insufficient vitamin B12 leads to pernicious anaemia, a disease that results in nervous disturbances and low production of red blood cells, though this is mostly due to lack of the gastric glycoprotein (intrinsic factor) which complexes with the vitamin to facilitate its absorption. Traditionally, daily consumption of raw liver was used to counteract the problem. Cyanocobalamin (or preferably hydroxocobalamin, which has a longer lifetime in the body) may be administered orally or by injection to counteract deficiencies. Both agents are converted into coenzyme B12 in the body. Coenzyme B12 is a cofactor for a number of metabolic rearrangements, such as the conversion of methylmalonyl-CoA into succinyl-CoA in the oxidation of fatty acids with an odd number of carbon atoms, and for methylations, such as in the biosynthesis of methionine from homocysteine.

H2NOC

CONH2

H2NOC

N R

N

CONH2

Co+

H

N

H2NOC

N

R = CN, cyanocobalamin (vitamin B12) R = OH, hydroxocobalamin (vitamin B12a) R = H2O, aquocobalamin (vitamin B12b) R = NO2, nitritocobalamin (vitamin B12c) R = Me, methylcobalamin (methyl vitamin B12) NH2 N

CONH2

N

R=

N

O OH O P O HO

HO

OH

5′-deoxyadenosylcobalamin (coenzyme B12)

HO N O

NH

N

NH

N

N

N

corrin ring system Figure 2.30

N

O

O NH

N

N HN

porphyrin ring system

34 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 2.1 (continued) Vitamin H Vitamin H (biotin; Figure 2.29) is a water-soluble vitamin found in eggs, liver, kidney, yeast, cereals, and milk; it is also produced by intestinal microflora, so that dietary deficiency is rare. Deficiency can be triggered by a diet rich in raw egg white, which contains the protein avidin that binds biotin so tightly that it is effectively unavailable for metabolic use. This affinity disappears on cooking which denatures the avidin. Biotin deficiency leads to dermatitis and hair loss. The vitamin functions as a carboxyl carrier, binding CO2 via a carbamate link, then donating this in carboxylase reactions, e.g. carboxylation of acetyl-CoA to malonyl-CoA (see page 17), of propionyl-CoA to methylmalonyl-CoA (see page 55), and of pyruvate to oxaloacetate during gluconeogenesis.

ELUCIDATING BIOSYNTHETIC PATHWAYS A biosynthetic pathway is a sequence of chemical transformations; with few exceptions, each of these reactions is catalysed by an enzyme. Chemical modification of a substrate depends upon the binding properties conferred by a particular combination of functional groups in the constituent amino acids of the protein. As a result, enzymes tend to demonstrate quite remarkable specificity towards their substrates, and usually catalyse only a single transformation. The main thrust of biosynthetic research has been in delineating the sequence of reactions and characterizing the enzymes involved. However, as the knowledge base increases, it is becoming possible through gene methodology to manipulate these processes to provide modified or new chemicals in the search for medicinal agents. This book summarizes the current knowledge about selected groups of natural products. How this knowledge has been acquired is the result of decades of meticulous research, often achieved without the benefits of modern technology. Not all pathways are known with the same level of detail; those shown in the figures present a composite picture of information accumulated by different methodologies at different times. A brief outline of the methods used for the elucidation of biosynthetic pathways helps to emphasize how these have changed over the years and should assist in interpretation of these figures. Though the methods have changed, it is most reassuring to realize that essentially all of the information gathered in the earlier studies has been confirmed correct by the newer techniques. Any biosynthetic study begins with speculation, comparing structurally related compounds from the same or different organisms, and suggesting possible interrelationships based on known chemical reactions, i.e. application of ‘paper chemistry’. In order to test the various suggestions, it is necessary to demonstrate that one compound is converted into another in a living organism, or, eventually, by an enzyme. This usually entails feeding an isotopically labelled compound to a plant, microorganism, animal

tissue, etc, that produces the required metabolite. For success, it is an absolute requirement that the organism is actively synthesizing the compound during the feeding period. The metabolite is then isolated, purified, and analysed to detect presence of the isotope. The formation of a labelled product is cautiously interpreted as a demonstration of the precursor–product relationship, though it should also be confirmed that the position of labelling in the product is the same as that in the precursor; organisms can degrade an introduced chemical to smaller portions and utilize it in their general metabolism. Isotope detection methods need to be sensitive, since only small amounts of the added precursor may penetrate to the site of synthesis, and much may be metabolized on the way. Incorporations in microorganisms are typically very much higher than in plants. A realistic pathway can then be proposed by the combined results from a series of feeding experiments with different labelled substrates. The biosynthetic origins of the animal sterol cholesterol are described in detail in Chapter 5 (see page 248), but for the present, can be used to illustrate some of the methodologies. In the early studies exploring the role of acetic acid in cholesterol biosynthesis in rats and rat liver, the isotope used was deuterium and mass spectroscopic detection was employed. When the radioactive isotopes carbon-14 (14 C) and tritium (3 H) became available, the level of sensitivity was increased significantly. In due course, it was established that all carbon atoms in cholesterol are derived from acetic acid, as shown in Figure 2.31. This necessitated systematic chemical degradation of the labelled cholesterol to allow each carbon to be separately assessed for labelling, a painstaking and outstanding achievement. Cholesterol was found to originate from acetate, incorporating 15 methyl and 12 carboxyl carbon atoms. Actually, cholesterol biosynthesis requires 18 acetate units, and these numbers indicate loss of three methyl groups and six carboxyl groups. This is because an early precursor, mevalonic acid, is formed from three acetate groups (as acetyl-CoA), and six mevalonate groups

Secondary Metabolism: The Building Blocks and Construction Mechanisms C5 isoprene unit

OH

∗

∗

OH

O

HO2C OH

O

∗

OH mevalonic acid (MVA)

∗ ∗

HO

∗

∗ ∗

∗

35

∗

∗

∗

∗ cholesterol

carbon lost

Figure 2.31 are used in producing squalene, a C30 precursor of cholesterol (C27 ). It had initially been shown that feeding of squalene, found in shark liver, increased the cholesterol content of animal tissues. Mevalonate is the source of the biosynthetic C5 isoprene unit that is used for steroid biosynthesis. The cyclization of squalene (as the oxide) provides the tetracyclic ring system of lanosterol; the process also involves some structural rearrangements involving methyl and hydride transfers, typical of carbocation chemistry, as briefly outlined in Figure 2.32. In due OH HO2C

OH HO2C

OH T H

OH H T

[2-14C,(4R)-3H]MVA 3H/14C

course, lanosterol (C30 ) is converted into cholesterol (C27 ) with loss of three carbon atoms. The characteristic β-radiations from 14 C and 3 H have different energies and can be measured separately in a mixed sample. This allowed multiple-labelling studies, tracking the retention or loss of hydrogen. To establish loss of hydrogen, it is pointless feeding a substrate to and then detecting no isotope in the product; this could mean no incorporation of the precursor. Instead, the carbon isotope is used as the reference label, and retention

[2-14C,(4S)-3H]MVA T

= 1:1 all 3H lost T

T T

T

T T

T

T

T

T

squalene

H/14C = 6:6

T O

squalene oxide

3

cyclizations

T

T

T

T

T

T

T

T H 3

HO

H

H

H cholesterol 3H/14C

Figure 2.32

= 3:5

T

3

HO

T

HO

T lanosterol 3H/14C

= 5:6

T

T protosteryl cation 3H/14C

= 6:6

T

36 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition or loss of hydrogen (the indicator label) is established by comparison of the 3 H/14 C ratio. Establishing the position of tritium labelling is often easier than employing full degradation; simple chemical modification, e.g. oxidation of an alcohol to a ketone, might remove a hydrogen atom and allow the site of labelling to be deduced. The multiple-labelling technique provided an approach not only to the fine detail of cholesterol formation, but also to the mechanistic aspects for the mevalonate to squalene conversion, and the formation of the biosynthetic isoprene unit. In particular, the stereochemical aspects could be established by feeding experiments with a series of stereospecifically labelled mevalonates. Thus, in Figure 2.32, it can be seen that the 4-pro-S hydrogen is lost, whilst the 4-pro-R hydrogen is retained through subsequent reactions. The predicted rearrangements involving hydride migrations were also confirmed. A small detail in Figure 2.32 is the loss of tritium from C-3 in going from lanosterol to cholesterol; this was interpreted correctly as indicating the conversion probably involved a C-3 carbonyl group. Labelling studies employing the non-radioactive isotope carbon-13 (13 C) have made a huge impact on biosynthetic methodology. This has generally eliminated the need for degradations, and 13 C has effectively displaced the use of 14 C altogether. The natural abundance of 13 C is 1.1%, and modern NMR spectrometers can detect this in relatively small samples of a compound. Hence, any biosynthetic enrichment can be detected simply by an enhancement of the natural abundance signal. Furthermore, the position of labelling is immediately established from the spectrum via the assignment of the enhanced signal. The results of Figure 2.31 would be obtained in hours instead of years. The use of double-labelled precursors has made even greater impact. Most frequently,

OH O [1,2-13C2]acetate

researchers have employed [1,2-13 C2 ]acetate, a compound in which each carbon atom is >99% 13 C. When this substrate is incorporated intact into a metabolite, the C2 unit can be detected in the 13 C NMR spectrum because of spin–spin coupling between the two adjacent nuclides. Instead of enhanced signals, new doublets appear flanking the natural abundance signal. Statistically, it is unlikely that labelled molecules of precursor will be incorporated consecutively into a single molecule of product, so additional couplings that might complicate the spectrum will be negligible. Any acetate precursor that is cleaved during biosynthesis will end up enhancing a natural abundance signal, and not show any coupling. Cholesterol derived from [13 C2 ]acetate would display couplings/enrichments as illustrated in Figure 2.33. There also exists the opportunity to employ [13 CD3 ]acetate and to detect the deuterium by isotope-induced shifts in the 13 C spectrum. Note that [14 C,3 H]mevalonate in the previous paragraph refers to a mixture of 14 C-labelled and 3 H-labelled substrates; this is quite different from the double-labelled substrate considered here, and the NMR coupling observed is a consequence of the labelled atoms being adjacent in the same molecule. Experiments with simple precursors such as acetate and mevalonate have their limitations, and further details of the pathway may require synthesis of more complex substrates in labelled form, and their observed transformation into final products. Thus, the biosynthesis of cholesterol from acetyl-CoA requires over 30 different enzymes; the transformation of lanosterol into cholesterol alone is a 19-step process requiring nine enzymes, some being used more than once. Often, the fine detail of the pathway can only be established via isolation and detailed study of the enzymes themselves. Approaches to enzymes have

OH HO2C

H

OPP

OH mevalonic acid (MVA)

isopentenyl PP (IPP) H HO

intact acetate C2 unit

H

labelled carbon from cleaved C2 unit

H H

H

HO cholesterol

Figure 2.33

H protosteryl cation

H

Secondary Metabolism: The Building Blocks and Construction Mechanisms changed dramatically in recent years. Initially, it was necessary to obtain a crude protein extract from a tissue known to be capable of synthesizing the molecule under investigation. The protein extract was then subjected to extensive fractionation to provide a portion that contained the appropriate activity. Since enzymes only catalyse a limited chemical change, this necessitated supplying the extract with a substrate, usually in labelled form to facilitate assays, together with essential cofactors as required, and observing its conversion into the next intermediate on the pathway. Enzyme isolation and fractionation was tedious, and the amount of protein obtained was often very small indeed, limiting subsequent studies. Recent rapid progress in enzyme production is the result of significant advances in genetic techniques. A gene is a segment of DNA that contains the information necessary for the synthesis of a particular protein/enzyme. Its identification initially required knowledge of the peptide sequence determined from the purified enzyme. Now, it is possible to search for likely genes in DNA sequences, produce them synthetically, and to express them in a suitable bacterium or yeast; to avoid complications with the normal biosynthetic machinery, this is usually different from the source organism. Recombinant proteins can then be tested for the expected enzyme activity. The search is facilitated by published gene sequences for similar enzymes, or by characteristic sequences that can be assigned to a particular class of enzyme, usually by the need to bind a specific cofactor, e.g. NADPH, SAM, cytochrome P-450, etc. In

some organisms, especially bacteria and fungi, a group of genes involved in secondary metabolite biosynthesis may lie in close proximity as gene clusters. This again makes identification considerably easier, and can provide further information as the roles of all the genes are clarified. It should be appreciated that, although enzymes from different sources may catalyse the same reaction on the same substrate, the proteins may not have the same amino acid sequence, though they are likely to be identical or similar for most of sequence, especially the functional part. Hence, relevant gene sequences are also likely to vary, but, again, there is going to be a high proportion of identity or similarity. These techniques allow accumulation of significant amounts of enzymes for mechanistic studies of the pathways. Various compounds can be tested as potential inhibitors or alternative substrates. For example, the stereochemistry of the side-chain at C-17 of the protosteryl cation in cholesterol biosynthesis was revised from 17α to 17β following studies with the enzyme oxidosqualene:lanosterol cyclase (Figure 2.34). When the oxygen analogue 20-oxa-2,3-oxidosqualene was used as substrate for the enzyme, the protosteryl cation analogue formed decomposed due to the presence of an oxygen function adjacent to the cationic centre. The stable ketone product obtained possessed a 17β-side-chain. Modified genes can be synthesized to produce new proteins with specific changes to amino acid residues, thus shedding more light on the enzyme’s mode of action. Specific genes can be damaged or deleted to prevent

H

oxidosqualene: lanosterol cyclase

H 17

lanosterol

H HO

O 2,3-oxidosqualene

H protosteryl cation

O

O H oxidosqualene: lanosterol cyclase

O H

H

O 20-oxa-2,3-oxidosqualene

Figure 2.34

H 17

H HO

37

H oxaprotosteryl cation

H HO

H

protosterol methylketone

38 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition a particular enzyme being expressed in the organism. Genes from different organisms can be combined and expressed together so that an organism synthesizes abnormal combinations of enzyme activities, allowing production of modified products. However, an organism should not be viewed merely as a sackful of freely diffusible and always available enzymes; biosynthetic pathways are under sophisticated controls in which there may be restricted availability or localization of enzymes and/or substrates. Enzymes involved in the biosynthesis of many important secondary metabolites may be grouped together as enzyme complexes, or may form part of a multifunctional protein. Nevertheless, biosynthesis has indeed entered a new era; it has moved on from an information-gathering exercise to a technology in which the possibilities appear to be limitless.

FURTHER READING Organic Chemistry, Mechanism Dewick PM (2006) Essentials of Organic Chemistry for Students of Pharmacy, Medicinal Chemistry and Biological Chemistry. John Wiley & Sons, Ltd, Chichester. McMurry J and Begley T (2005) The Organic Chemistry of Biological Pathways. Roberts, Englewood, CO.

Natural Products, Biosynthesis Bugg TDH (2001) The development of mechanistic enzymology in the 20th century. Nat Prod Rep 18, 465–493.

Stanforth SP (2006) Natural Product Chemistry at a Glance. Blackwell, Oxford. Thomas R (2004) Biogenetic speculation and biosynthetic advances. Nat Prod Rep 21, 224–248.

Vitamins Amadasi A, Bertoldi M, Contestabile R, Bettati S, Cellini B, di Salvo ML, Voltattorni CB, Bossa F and Mozzarelli A (2007) Pyridoxal 5 -phosphate enzymes as targets for therapeutic agents. Curr Med Chem 14, 1291–1324. Bartlett MG (2003) Biochemistry of the water soluble vitamins: a lecture for first year pharmacy students. Am J Pharm Educ 67, 1–7. Begley TP (2006) Cofactor biosynthesis: an organic chemist’s treasure trove. Nat Prod Rep 23, 15–25. Begley TP, Chatterjee A, Hanes JW, Hazra A and Ealick SE (2008) Cofactor biosynthesis – still yielding fascinating new biological chemistry. Curr Opin Chem Biol 12, 118–125. Kluger R and Tittmann K (2008) Thiamin diphosphate catalysis: enzymic and nonenzymic covalent intermediates. Chem Rev 108, 1797–1833. R´ebeill´e F, Ravanel S, Marquet A, Mendel RR, Smith AG and Warren MJ (2007) Roles of vitamins B5 , B8 , B9 , B12 and molybdenum cofactor at cellular and organismal levels. Nat Prod Rep 24, 949–962. Roje S (2007) Vitamin B biosynthesis in plants. Phytochemistry 68, 1904–1921. Smith AG, Croft MT, Moulin M and Webb ME (2007) Plants need their vitamins too. Curr Opin Plant Biol 10, 266–275.

3

THE ACETATE PATHWAY: FATTY ACIDS AND POLYKETIDES

Polyketides constitute a large class of natural products grouped together on purely biosynthetic grounds. Their diverse structures can be explained as being derived from poly-β-keto chains, formed by coupling of acetic acid (C2 ) units via condensation reactions, i.e. nCH3 CO2 H → —[CH2 CO]n — Included in such compounds are the fatty acids, polyacetylenes, prostaglandins, macrolide antibiotics and many aromatic compounds, e.g. anthraquinones and tetracyclines. The formation of the poly-β-keto chain could be envisaged as a series of Claisen reactions, the reverse of which are involved in the β-oxidation sequence for the metabolism of fatty acids (see page 18). Thus, two molecules of acetyl-CoA could participate in a Claisen condensation giving acetoacetyl-CoA, and this reaction could be repeated to generate a poly-β-keto ester of appropriate chain length (Figure 3.1). However, a study of the enzymes involved in fatty acid biosynthesis showed this simple rationalization could not be correct and that a more complex series of reactions was operating. It is now known that fatty acid biosynthesis involves initial carboxylation of acetyl-CoA to malonyl-CoA, a reaction involving ATP, CO2 (as bicarbonate, HCO3 − ), and the coenzyme biotin as the carrier of CO2 (see page 17). The conversion of acetyl-CoA into malonyl-CoA increases the acidity of the α-hydrogen atoms, thus providing a better nucleophile for the Claisen condensation. In the biosynthetic sequence, no acylated malonic acid derivatives are produced, and no label from [14 C]bicarbonate is incorporated, so the carboxyl group introduced into malonyl-CoA is simultaneously lost by a decarboxylation reaction during the Claisen condensation

(Figure 3.1). Accordingly, the carboxylation step helps to activate the α-carbon and facilitate Claisen condensation, and the carboxyl is immediately removed on completion of this task. An alternative rationalization is that decarboxylation of the malonyl ester is used to generate the acetyl enolate anion without any requirement for a strong base. The pathways to fatty acids, macrolides, and aromatic polyketides branch early. The chain extension process of Figure 3.1 continues for aromatics, generating a highly reactive poly-β-keto chain that is stabilized by association with groups on the enzyme surface until chain assembly is complete and cyclization reactions occur. However, for fatty acids, the carbonyl groups are reduced before attachment of the next malonate group. Partial reduction processes, leading to a mixture of methylenes, hydroxyls, and carbonyls, are characteristic of macrolides (see page 68). FATTY ACID SYNTHASE: SATURATED FATTY ACIDS The processes of fatty acid biosynthesis are well studied and are known to be catalysed by the enzyme fatty acid synthase (FAS). FASs from various organisms show significant structural differences. In animals, FAS is a large multifunctional protein with seven discrete functional domains, providing all of the catalytic activities required. All domains are on a single polypeptide, encoded by a single gene, though the enzyme exists as a homodimer and requires both units for activity. Fungal FAS is also a multifunctional enzyme, but the seven component activities are distributed over two non-identical polypeptides α and β, and the enzyme is an α6 β6 dodecamer. The

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

40 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Claisen reaction: acetyl-CoA acetyl-CoA

acetyl-CoA O

O CH3

SCoA

acetyl-CoA

Claisen reaction

CH3

O

O SCoA

CH3

Claisen reaction

acetoacetyl-CoA

O

O

SCoA n poly-β-keto ester

Claisen reaction: malonyl-CoA Claisen reaction

O CH3

SCoA O

O CH3

SCoA O

O SCoA O

CO2

CH3

malonyl-CoA CO2 CH3

repeat of Claisen reaction

SCoA

O alternatively, enzyme generates transient enolate anion O

O

SCoA

acetoacetyl-CoA

O malonyl-CoA

H nucleophilic attack on carbonyl with simultaneous loss of CO2

O

O

O

SCoA n poly-β-keto ester

O SCoA

CH2

SCoA

O H

B Enz

Figure 3.1 multifunctional protein systems in animals and fungi are termed type I FASs. Bacteria and plants possess a type II FAS that is an assembly of separable enzymes, encoded by seven different genes. Nevertheless, all of the different FAS systems perform effectively the same task employing the same mechanisms. Acetyl-CoA and malonyl-CoA themselves are not involved in the condensation step: they are converted into enzyme-bound thioesters, the malonyl ester by means of an acyl carrier protein (ACP) (Figure 3.2). The Claisen reaction follows giving acetoacetyl-ACP (β-ketoacyl-ACP; R = H), which is reduced stereospecifically to the corresponding β-hydroxy ester, consuming NADPH in the reaction. Then follows elimination of water giving the E (trans) α,β-unsaturated ester. Reduction of the double bond again utilizes NADPH and generates a saturated acyl-ACP (fatty acyl-ACP; R = H) which is two carbon atoms longer than the starting material. This can feed back into the system, condensing again with malonyl-ACP and going through successive reduction, dehydration and reduction steps, gradually increasing the chain length by two carbon atoms for each cycle until the required chain length is obtained. At that point, the fatty acyl chain can be released as a fatty acyl-CoA or as the free acid. Fungi typically produce the CoA esters by a reversal of the loading reaction, whilst mammals release the free acid by the

action of a thioesterase (TE). The chain length actually elaborated is probably controlled by the specificity of the TE enzyme. The FAS protein is known to contain an ACP binding site, and also an active-site cysteine residue in the ketosynthase (KS) domain. Acetyl and malonyl groups are successively transferred from coenzyme A esters and attached to the thiol groups of Cys and ACP respectively by the same malonyl/acetyl transferase (MAT; Figure 3.3). The Claisen condensation occurs (KS), and the processes of reduction (ketoreductase, KR), dehydration (dehydratase, DH), and reduction (enoyl reductase, ER) then occur whilst the growing chain is attached to ACP. The ACP carries a phosphopantetheine group exactly analogous to that in coenzyme A, and this provides a long flexible arm, enabling the growing fatty acid chain to reach the active site of each enzyme in the complex, allowing the different chemical reactions to be performed without releasing intermediates from the enzyme (compare polyketide synthesis on page 70 and peptide synthesis on page 438). The sequence of enzyme activities along the protein chain of the enzyme complex does not correspond with the order in which they are employed (see Figure 3.4). Then the chain is transferred to the thiol of Cys, and the process can continue. Making the process even more efficient, the dimeric animal FAS contains

The Acetate Pathway: Fatty Acids and Polyketides O

load extender unit SCoA

O

ACP−SH

CO2H

ACP: acyl carrier protein SACP

CO2H

E1

malonyl-CoA

load starter unit

malonyl-ACP

O RCH2

Claisen reaction E2 H OH O RCH2 R

O SACP

E2 elimination E4 of H2O

– H2O

CH3

E1

SCoA

acetyl-CoA

transfer acyl group

RCH2 SACP E3 β-ketoacyl-ACP stereospecific reduction of carbonyl

Enz−SH

O

each turn of the cycle extends the chain length of the acyl group by two carbons

O NADPH

E RCH2

SEnz

O

O

Enz−SH

acyl−enzyme thioester

NADPH

β-hydroxyacyl-ACP

41

SACP

α,β-unsaturated acyl-ACP

RCH2

E5 reduction of double bond

fatty acyl-ACP HSCoA

Enzyme activities of mammalian fatty acid synthase E1: malonyl/acetyl transferase (MAT; malonyl-CoA/acetyl-CoA-ACP-transacylase) E2: ketosynthase (KS; β-ketoacyl synthase) E3: ketoreductase (KR; β-ketoacyl reductase) E4: dehydratase (DH) E5: enoyl reductase (ER; β-enoyl reductase) E6: thioesterase (TE)

SACP

E6

O RCH2

H2O O

SCoA fatty acyl-CoA

RCH2

OH fatty acid

Figure 3.2 two catalytic centres and is able to generate two growing chains at the same time. The monomeric subunits are also arranged so that, although each monomer can catalyse the full set of reactions, there also exists the potential for utilizing domains on the other subunit for the initial reactions of substrate loading (MAT) and Claisen condensation (KS). Although fungal FAS is also a type I multifunctional protein, there are some differences compared with the mammalian enzyme. Thus, the functional domains are arranged on two separate polypeptides (Figure 3.4), and the complex is a dodacamer rather than a dimer. Acetyl-CoA and malonyl-CoA are loaded onto the system by separate enzyme activities, namely acetyl transferase (AT) and malonyl/palmitoyl transferase (MPT), the latter also being involved in releasing the product palmitoyl-CoA from the ACP. A further enzyme activity is also included, phosphopantetheinyl transferase (PPT), which is required for activating the ACP by inserting the phosphopantetheine arm. The dissociated FAS II system found in most bacteria and plants consists of a series of discrete

proteins, each of which catalyses an individual reaction of the fatty acid biosynthetic pathway. In some cases, two or more enzymes are able to perform the same chemical reaction, but have differing substrate specificities. For example, three distinct KS activities with characteristic chain-length specificities have been identified in plants. One condenses acetyl-CoA with malonyl-ACP, one is responsible for further elongation to C16 , and a third one elongates C16 to C18 . This allows both C16 and C18 metabolites to be tapped off for different functions. Unusually, the tuberculosis-causing bacillus Mycobacterium tuberculosis is known to possess both FAS I and FAS II systems. It uses FAS I to produce acyl chains in the range C14 –C16 , and FAS II to elongate these structures to mycolic acids, very long-chain fatty acids with 24–56 carbon atoms that are components of the mycobacterial cell wall. Because of the fundamental differences in mammalian FAS I and bacterial FAS II, there is considerable potential for exploiting these differences and developing selective inhibitors of fatty acid synthesis as antibacterial agents. Indeed, this approach has been

42 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O

loading SH

O

acetyl-CoA malonyl-CoA

SH

S

S

Cys

Claisen reaction ketosynthase (KS)

O O SH

S

transfer

O

reduction dehydration reduction

SH

S Cys

ACP

ACP

R

S

R

O S

O

ACP

ketoreductase (KR) dehydratase (DH) enoyl reductase (ER)

ACP

O growing fatty acyl chain

O

SH

Cys

bonding through phosphopantetheine

H

ACP

malonyl/acetyl transferase (MAT)

fatty acid synthase

Cys

O

Cys ACP

S

O

CH3

O

O

P N H

cysteamine

O

N H

OH pantothenic acid

O OH

CH2

Ser

ACP

pantetheine bound to serine group of ACP through phosphate

phosphopantetheine

Figure 3.3

domain organization: mammalian FAS ⎯ KS ⎯ MAT ⎯ DH ⎯ ER ⎯ KR ⎯ ACP ⎯ TE ⎯ domain organization: fungal FAS α

⎯ ACP ⎯ KR ⎯ KS ⎯ PPT ⎯

β

⎯ AT ⎯ ER ⎯ DH ⎯ MPT ⎯

ACP: acyl carrier protein AT: acetyl transferase DH: dehydratase ER: enoyl reductase KR: ketoreductase KS: ketosynthase MAT: malonyl/acetyl transferase MPT: malonyl/palmitoyl transferase PPT: phosphopantetheinyl transferase TE: thioesterase

Figure 3.4 successful with agents such as isoniazid (antituberculosis) and triclosan (general biocide), both inhibitors of bacterial ER. Considerable effort is being invested in targeting FAS II in Plasmodium falciparum, the causative agent of malaria. The combination of one acetate starter unit with seven malonate extender units would give the C16 fatty acid, palmitic acid, and with eight malonates the C18 fatty acid, stearic acid (Figure 3.5). Note that the two carbon atoms at the head of the chain (methyl end) are provided by acetate, not malonate, whilst the remainder are derived from malonate. However, malonate itself is produced by carboxylation of acetate. This means that all carbon atoms in the fatty acid originate from acetate, but malonate will

only provide the C2 chain extension units and not the C2 starter group. The linear combination of acetate C2 units as in Figure 3.2 explains why the common fatty acids are straight-chained and possess an even number of carbon atoms. Natural fatty acids may contain from 4 to 30, or even more, carbon atoms, the most abundant being those with 16 or 18 carbon atoms. Some naturally occurring fatty acids are shown in Figure 3.6. The rarer fatty acids containing an odd number of carbon atoms typically originate from incorporation of a different starter unit, e.g. propionic acid, or can arise by loss of one carbon from an even-numbered acid. Other structures (see page 53) can arise by utilizing different starter units and/or different extender units.

The Acetate Pathway: Fatty Acids and Polyketides O

O SCoA acetyl-CoA

O

O SCoA acetyl-CoA

SCoA CO2H malonyl-CoA

SCoA CO2H malonyl-CoA

7×

8×

CO2H starter unit

43

palmitic acid (16:0)

extender units

CO2H starter unit

stearic acid (18:0)

extender units

Figure 3.5

Fatty acids are mainly found in ester combination with glycerol in the form of triglycerides (Figure 3.7). These materials are called fats or oils, depending on whether they are solid or liquid at room temperature. If all three esterifying acids are the same, then the triglyceride is termed simple, whereas a mixed triglyceride is produced

if two or more of the fatty acids are different. Most natural fats and oils are composed largely of mixed triglycerides. In this case, isomers can exist, including potential optical isomers, since the central carbon will become chiral if the primary alcohols of glycerol are esterified with different fatty acids. In practice, only one of each pair

Common naturally occurring fatty acids

Saturated butyric

4:0

caproic *

6:0

caprylic *

8:0

capric *

10:0

lauric

12:0

myristic

14:0

palmitic

16:0

stearic

18:0

arachidic

20:0

behenic

22:0

lignoceric

24:0

cerotic

26:0

montanic

28:0

melissic

30:0

Abbreviations:

CO2H

Number of carbon atoms

CO2H

Position of double bonds

CO2H

18:2 (9c,12c)

CO2H

Stereochemistry of double bonds (c = cis/Z; t = trans/E)

CO2H

Number of double bonds

CO2H CO2H

CO2H CO2H CO2H

* To avoid confusion, systematic nomenclature (hexanoic, octanoic, decanoic) is recommended

Figure 3.6 (continued overleaf)

CO2H CO2H CO2H CO2H

44 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Unsaturated palmitoleic

CO2H

16:1 (9c)

oleic

CO2H

18:1 (9c)

cis-vaccenic

CO2H

18:1 (11c)

linoleic

CO2H

18:2 (9c,12c)

α-linolenic

CO2H

18:3 (9c,12c,15c)

γ-linolenic

CO2H

18:3 (6c,9c,12c)

gadoleic

CO2H

20:1 (9c)

gondoic

CO2H

20:1 (11c)

arachidonic

CO2H

20:4 (5c,8c,11c,14c)

eicosapentaenoic (EPA)

CO2H

20:5 (5c,8c,11c,14c,17c)

cetoleic

CO2H

22:1 (11c)

erucic

CO2H

22:1 (13c)

docosapentaenoic (DPA)

CO2H

22:5 (7c,10c,13c,16c,19c)

docosahexaenoic (DHA) nervonic

CO2H

22:6 (4c,7c,10c,13c,16c,19c)

CO2H

24:1 (15c)

Figure 3.6 (continued) of enantiomers is formed in nature. Triglycerides are produced predominantly from glycerol 3-phosphate by esterification with fatty acyl-CoA residues, the phosphate being removed prior to the last esterification (Figure 3.7). The diacyl ester of glycerol 3-phosphate is also known as a phosphatidic acid, and is the basis of phospholipid structures. In these structures, the phosphate is also esterified with an alcohol, which is usually choline, ethanolamine, serine, or myo-inositol, e.g. phosphatidyl choline (Figure 3.8). Phospholipids are important structural components of cell membranes, and because of the polar and non-polar regions in their structure, they have detergent-like properties. They are also able to form liposomes, which have considerable potential as drug delivery systems. A particularly important natural phospholipid is platelet-activating factor (PAF; Figure 3.8), which resembles a phosphatidylcholine, though this compound possesses an ether linkage to a long-chain fatty alcohol, usually hexadecanol, rather than an ester linkage.

The central hydroxyl of glycerol is esterified, but to acetic acid rather than to a long-chain fatty acid. PAF functions at nanomolar concentrations, activates blood platelets, and contributes to diverse biological effects, including thrombosis, inflammatory reactions, allergies, and tissue rejection. Long-chain alcohols are reduction products from fatty acyl-CoA esters; they also feature in natural waxes. Waxes are complex mixtures of esters of long-chain fatty acids, usually C20 –C24 , with long-chain monohydric alcohols or sterols. The alcohol is esterified using a fatty acyl-CoA. UNSATURATED FATTY ACIDS Animal fats contain a high proportion of glycerides of saturated fatty acids and tend to be solids, whilst those from plants and fish contain predominantly unsaturated fatty acid esters and tend to be liquids. Some of the common naturally occurring unsaturated fatty acids are also included in Figure 3.6. A convenient shorthand

The Acetate Pathway: Fatty Acids and Polyketides O

O R1

R2

SCoA HO

E1

OP glycerol 3-P

SCoA

OCOR1

OH HO

OP

esterification with first fatty acyl-CoA

1-acylglycerol 3-P (lysophosphatidic acid)

OCOR1 E2 esterification with second fatty acyl-CoA

R2COO OP 1,2-diacylglycerol 3-P (phosphatidic acid) H2O

E3 E1: E2: E3: E4:

45

glycerol 3-phosphate acyltransferase (GPAT) lysophosphatidic acid acyltransferase (LPAT) phosphatidic acid phosphatase (PAP) diacylglycerol acyltransferase (DGAT) R2COO

hydrolysis of phosphate

O R3

OCOR1

SCoA

OCOR1 R2COO

E4

OCOR3 triacylglycerol (triglyceride)

OH

esterification with third fatty acyl-CoA

1,2-diacylglycerol

Figure 3.7

NMe3

R3 = OCOR1 R2COO

OH O

OR3

P O

phosphatidylcholine

NH3

phosphatidylethanolamine

CO2

phosphatidylserine

OH O

NH3 HO phosphatidyl-myo-inositol OH OH OH

OR MeCOO

O P

NMe3

O platelet-activating factor (PAF) e.g. R = (CH2)15CH3

OH

Figure 3.8 representation for fatty acids indicating chain length with number, position and stereochemistry of double bonds is also presented in Figure 3.6. A less systematic numbering starting from the methyl (the ω end) may also be encountered. Major groups of fatty acids are designated ω–3 (omega-3), ω–6 (omega-6), ω–9 (omega-9), etc. (or sometimes n–3, n–6, n–9), if there is a double bond that number of carbon atoms from the methyl terminus. This has some value in relating structures when an unsaturated fatty acid is biosynthetically elongated from the carboxyl end as during prostaglandin biosynthesis (see page 50). Double bonds at position 9 are common, but unsaturation can occur at other positions in the chain. Polyunsaturated fatty acids tend to have their double bonds in a non-conjugated array as a repeating unit —(CH CHCH2 )n —. In virtually all cases,

the stereochemistry of the double bond is Z (cis), thus introducing a ‘bend’ into the alkyl chain. This interferes with the close association and aggregation of molecules that is possible in saturated structures and helps to maintain the fluidity in oils and cellular membranes. Fats and oils represent long-term stores of energy for most organisms, being subjected to oxidative metabolism as required. Major oils which are produced commercially for use as foods, toiletries, medicinals, or pharmaceutical formulation aids are listed in Table 3.1. Typical fatty acid analyses are shown, though it must be appreciated that these figures can vary quite widely. For instance, plant oils show significant variation according to the climatic conditions under which the plant was grown. In colder climates, a higher proportion of polyunsaturated fatty acids is produced, so that the plant can maintain the fluidity

Prunus amygdalus var. dulcis, or var. amara (Rosaceae) Arachis hypogaea (Leguminosae/Fabaceae)

Almond

Borago officinalis (Boraginaceae)

cow Bos taurus (Bovidae)

Ricinus communis (Euphorbiaceae)

Cocos nucifera (Palmae/Arecaceae)

cod Gadus morrhua (Gadidae)

Gossypium hirsutum (Malvaceae)

Oenothera biennis (Onagraceae)

Borage (starflower)

Butterfat

Castor

Coconut

Cod-liver

Cottonseed

Evening primrose

Arachis (groundnut, peanut)

Source

Fixed oils and fats.a,b,c

Oil

Table 3.1

seed

seed

fresh liver

seed kernel

seed

milk

seed

seed

seed

Part used

24

15–36

50

65–68

35–55

2–5

28–35

45–55

40–55

Oil content (%)

linoleic (65–80), γ-linolenic (7–14), oleic (9), palmitic (7)

linoleic (33–58), palmitic (17–29), oleic (13–44), stearic (1–4)

lauric (43–53), myristic (15–21), palmitic (7–11), caprylic (5–10), capric (5–10), oleic (6–8), stearic (2–4) oleic (24), DHA (14), palmitic (11), EPA (6), palmitoleic (7), stearic (4), myristic (3)

oleic (35–72), linoleic (13–43), palmitic (7–16), stearic (1–7), behenic (1–5), arachidic (1–3) linoleic (38), γ-linolenic (23–26), oleic (16), palmitic (11) palmitic (29), oleic (28), stearic (13), myristic (12), butyric (4), lauric (3), caproic (2), capric (2), palmitoleic (2) ricinoleic (80–90), oleic (4–9), linoleic (2–7), palmitic (2–3), stearic (2–3)

oleic (62–86), linoleic (7–30), palmitic (4–9), stearic (1–2)

Typical fatty acid composition (%)

emollient base, purgative, soap manufacture; castor seeds contain the highly toxic, but heat-labile protein ricin (see page 435) soaps, shampoos; fractionated coconut oil containing only short- to medium-length fatty acids (mainly caprylic and capric) is a dietary supplement dietary supplement due to presence of EPA and DHA, plus vitamins A (see page 304) and D (see page 258); halibut-liver oil from halibut Hippoglossus vulgaris (Pleurnectideae) has similar properties and is used in the same way solvent for injections, soaps; cotton seeds also contain 1.1–1.3% gossypol (see page 220) and small amounts of cyclopropenoid fatty acids, e.g. sterculic and malvalic acids (see page 55) dietary supplement for γ-linolenic (gamolenic) acid content in treatment of premenstrual tension, breast pain, eczema

dietary supplement for γ-linolenic (gamolenic) acid content in treatment of premenstrual tension, breast pain, eczema food

food oil, emollient base

emollient base, toiletries, carrier oil (aromatherapy)

Uses, notes

46 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Linum usitatissimum (Linaceae)

Zea mays (Graminae/Poaceae) Olea europaea (Oleaceae) Elaeis guineensis (Palmae/Arecaceae)

Brassica napus (Cruciferae/ Brassicaceae)

Sesamum indicum (Pedaliaceae)

Linseed (flaxseed)

Maize (corn)

Rapeseed

Sesame

Palm kernel

Olive

pig Sus scrofa (Suidae)

various, including herring Clupea harengus (Clupeidae) (Europe) and menhaden Brevoortia spp. (Clupeidae) (N America) Lunaria annua (Cruciferae/ Brassicaceae)

Lard

Honesty

Fish

seed

seed

kernel

fruits

embryo

seed

abdominal fat

seed

whole fish – by-product of fishing and fish-meal industry

44–54

40–50

45–50

15–40

33–39

35–44

30–40

up to 20% (herring)

oleic (35–50), linoleic (35–50), palmitic (7–12), stearic (4–6)

linoleic (34–62), oleic (19–50), palmitic (8–19), stearic (0–4) oleic (56–85), palmitic (8–20), linoleic (4–20), stearic (1–4) lauric (40–52), myristic (14–18), oleic (9–16), palmitoleic (6–10), caprylic (3–6), capric (3–5), stearic (1–4), linoleic (1–3) erucic (30–60), oleic (9–25), linoleic (11–25), gadoleic (5–15), α-linolenic (5–12), palmitic (0–5)

oleic (45), palmitic (25), stearic (12), linoleic (10), palmitoleic (3) α-linolenic (30–60), oleic (39), linoleic (15), palmitic (7), stearic (4)

erucic (43), nervonic (25), oleic (24)

herring: cetoleic (7–30), oleic (9–25), gondoic (7–24), palmitic (10–19), EPA (4–15), palmitoleic (6–12), myristic (5–8), DHA (2–8), stearic (1–2)

(continued overleaf )

food oil, using varieties producing lower levels of erucic acid where the main components are now oleic (48–60%), linoleic (18–30%), α-linolenic (6–14%), and palmitic (3–6%) acids; erucic acid is known to accumulate and cause lesions in heart muscle; erucic acid is used as a plasticizer in PVC clingfilm food oil, soaps, solvent for injections, carrier oil (aromatherapy)

soaps; fractionated palm oil is a solid obtained by fractionation and hydrogenation and is used as a suppository base

liniments, dietary supplement for α-linolenic acid content; formerly the basis of paints, reacting with oxygen, polymerizing, and drying to a hard film food oil, dietary supplement, solvent for injections food oil, emollient base

nervonic acid is being investigated for the treatment of multiple sclerosis; the disease is characterized by a deficiency in nervonic acid food

dietary supplement for EPA and DHA (see page 49); refined and concentrated oils are produced containing higher amounts of EPA (15–30%) and DHA (10–20%)

The Acetate Pathway: Fatty Acids and Polyketides 47

Glycine max (Leguminosae/ Fabaceae)

sheep Ovis aries (Bovidae) cow Bos taurus (Bovidae)

Helianthus annuus (Compositae/ Asteraceae) Theobroma cacao (Sterculiaceae)

Soya (soybean)

Suet (mutton tallow)

Sunflower

kernel

seed

abdominal fat

abdominal fat

seed

Part used

35–50

22–36

18–20

Oil content (%)

oleic (35), stearic (35), palmitic (26), linoleic (3)

stearic (32), oleic (31), palmitic (27), myristic (6) oleic (48), palmitic (27), palmitoleic (11), stearic (7), myristic (3) linoleic (50–70), oleic (20–40), palmitic (3–10), stearic (1–10)

linoleic (44–62), oleic (19–30), palmitic (7–14), α-linolenic (4–11), stearic (1–5)

Typical fatty acid composition (%)

suppository base, chocolate manufacture; theobroma oil (cocoa butter) is a solid

food oil, carrier oil (aromatherapy)

foods

food oil, dietary supplement, carrier oil (aromatherapy); soya oil contains substantial amounts of the sterols sitosterol and stigmasterol (see page 255) foods

Uses, notes

particularly seeds and fruits, and the oil is extracted by cold or hot expression, or less commonly by solvent extraction with hexane. The crude oil is then refined by filtration, steaming, neutralization to remove free acids, washing and bleaching as appropriate. Many food oils are then partially hydrogenated to produce semi-solid fats. Animal fats and fish oils are usually extracted by steaming, the higher temperature deactivating enzymes which would otherwise begin to hydrolyse the glycerides. c Oils and fats feature as important food components and cooking oils, some 80% of commercial production being used as human food, whilst animal feeds account for another 6%. Most of the remaining production is used as the basis of soaps, detergents, and pharmaceutical creams and ointments. A number of oils are used as diluents (carrier or base oils) for the volatile oils employed in aromatherapy.

b The term fat or oil has no precise significance, merely describing whether the material is a solid (fat) or liquid (oil) at room temperature. Most commercial oils are obtained from plant sources,

fatty acids are shown in Figure 3.6 (see page 43, 44)

a The oil yields and fatty acid compositions given in the above table are typical values, and can vary widely. The quality of an oil is determined principally by its fatty acid analysis. Structures of the

Theobroma

Suet (beef tallow)

Source

(continued)

Oil

Table 3.1

48 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

The Acetate Pathway: Fatty Acids and Polyketides of its storage fats and membranes. The melting points of these materials depend on the relative proportions of the various fatty acids, reflecting primarily the chain length and the amount of unsaturation in the chain. Saturation and increasing chain length in the fatty acids gives a more solid fat at room temperature. Thus, butterfat and cocoa butter (theobroma oil) contain a relatively high proportion of saturated fatty acids and are solids. Palm kernel and coconut oils are both semi-solids having a high concentration of the saturated C12 acid lauric acid. A characteristic feature of olive oil is its very high oleic acid (18:1) content, whilst rapeseed oil possesses high concentrations of long-chain C20 and C22 fatty acids, e.g. erucic acid (22:1). Typical fatty acids in fish oils have high unsaturation and also long chain lengths, e.g. eicosapentaenoic acid (EPA) (20:5) and docosahexaenoic acid (DHA) (22:6) in cod liver oil. Unsaturated fatty acids can arise by more than one biosynthetic route, but in most organisms the common mechanism is by desaturation of the corresponding alkanoic acid, with further desaturation in subsequent steps. Most eukaryotic organisms possess a 9 -desaturase enzyme that introduces a cis double bond into a saturated fatty acid, requiring O2 and NADPH or NADH cofactors. The mechanism of desaturation does not involve any intermediates hydroxylated at C-9 or C-10, and the requirement for O2 is as an acceptor at the end of an electron transport chain. A stearoyl (C18 ) thioester is the usual substrate giving an oleoyl derivative (Figure 3.9), coenzyme A esters being utilized by animal and fungal enzymes, and ACP esters by plant systems. In some systems, desaturation may take place on fatty acids bound as lipids or phospholipids.

R = ACP in plants R = CoA in animals, fungi H

O

H

SR

E1 E2

O2 NADPH

stearic 18:0 O

9 10

SR oleic 18:1 (9c)

E1: stearoyl-ACP Δ9-desaturase E2: stearoyl-CoA Δ9-desaturase

Figure 3.9

49

The position of further desaturation then depends very much on the organism. Non-mammalian enzymes tend to introduce additional double bonds between the existing double bond and the methyl terminus, e.g. oleic acid → linoleic acid → α-linolenic acid (Figure 3.10). Animals introduce new double bonds towards the carboxyl group. They also lack the 12 and 15 desaturase enzymes, so, although linoleic acid and α-linolenic acid are necessary for the synthesis of polyunsaturated acids that lead to prostaglandins (see page 59) and leukotrienes (see page 64), animals must obtain these materials in the diet, mainly from plants. 6 -Desaturation (towards the carboxyl) of linoleic acid leads to γ-linolenic acid, whilst analogous desaturation of α-linolenic acid gives stearidonic acid. Prostaglandins are derived from C20 polyunsaturated fatty acid precursors, so addition of two extra carbon atoms is required. This is achieved by an elongase enzyme acting upon γ-linolenic acid, giving dihomo-γ-linolenic acid (8,11,14 -eicosatrienoic acid). The additional two carbon atoms derive from malonate, by the usual chain extension mechanism. Dihomo-γ-linolenic acid is the precursor of prostaglandins in the ‘one’ series; the ‘two’ series derives from arachidonic acid (5,8,11,14 -eicosatetraenoic acid), formed by further 5 -desaturation, again towards the carboxyl terminus. The ‘three’ series of prostaglandins are derivatives of Δ5,8,11,14,17 -eicosapentaenoic acid (EPA). This is an analogue of arachidonic acid formed from α-linolenic acid via stearidonic acid with a similar chain extension process and 5 -desaturation. Further chain extension of EPA gives docosapentaenoic acid (DPA), and then 4 -desaturation gives DHA. DHA is vital for proper visual and neurological development in infants, and deficiency has been associated with cognitive decline and the onset of Alzheimer’s disease in adults. Thus, a range of metabolites necessary for good health, including prostaglandins, leukotrienes, and these long-chain polyunsaturated fatty acids, are produced from the plant fatty acids linoleic acid and α-linolenic acid, which have to be obtained in the diet. Accordingly, these plant fatty acids are referred to as ‘essential fatty acids’ (EFAs). Further, since beneficial fatty acids, including α-linolenic acid, EPA, DPA, and DHA, have a double bond three carbon atoms from the methyl end of the chain, they are grouped together under the term ω–3 fatty acids (omega-3 fatty acids). Marine fish represent a major source of the nutritionally relevant longer chain fatty acids, especially EPA and DHA, which are not found in seed oils of higher plants. Regular consumption of oily fish, e.g. herring, tuna, mackerel, or use of fish oil supplements, is claimed to reduce the risk of heart attacks and atherosclerosis. Fish obtain most of

50 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

PLANT METABOLISM

desaturation towards methyl terminus

desaturation towards methyl terminus

6

CO SACP

plants fungi

plants fungi

CO SACP

CO SACP E2

E1 12

oleic 18:1 (9c)

α-linolenic 18:3 (9c,12c,15c)

linoleic 18:2 (9c,12c)

MAMMALIAN METABOLISM

desaturation towards carboxyl terminus

E3

E3

CO SCoA

CO SCoA

γ-linolenic 18:3 (6c,9c,12c) chain extension by Claisen E4 reaction with malonate; chain length increased by two carbons

+ C2 (malonate) 5

prostaglandins 1-series

stearidonic 18:4 (6c,9c,12c,15c) E4

+ C2 (malonate)

CO SCoA

CO SCoA

dihomo-γ-linolenic 20:3 (8c,11c,14c)

eicosatetraenoic 20:4 (8c,11c,14c,17c) desaturation towards carboxyl terminus

E5

E5 CO SCoA

CO SCoA

prostaglandins 2-series

eicosapentaenoic (EPA) 20:5 (5c,8c,11c,14c,17c)

arachidonic 20:4 (5c,8c,11c,14c) prostaglandins 3-series

E6

+ C2 (malonate)

4

CO SCoA

CO SCoA

E7 docosahexaenoic (DHA) 22:6 (4c,7c,10c,13c,16c,19c) E1: Δ12-desaturase E2: Δ15-desaturase

Figure 3.10

E3: Δ6-desaturase E4: C18 elongase

desaturation towards carboxyl terminus

E5: Δ5-desaturase E6: C20 elongase

E7: Δ4-desaturase

docosapentaenoic (DPA) 22:5 (7c,10c,13c,16c,19c) Note: the names given are for the appropriate fatty acid; the structures shown are the thioesters involved in the conversions

The Acetate Pathway: Fatty Acids and Polyketides these fatty acids by consumption of marine microalgae, which are considered to be the primary producers. It has also been found that these microalgae utilize another approach to synthesize unsaturated fatty acids, involving a multifunctional complex analogous to polyketide synthases (PKSs; see page 67) and a sequence that does not require desaturases and elongases. Some of these microalgae, e.g. Crypthecodinium cohnii and Schizochytrium spp., are currently exploited for the commercial production of DHA-enriched oils, which are also more palatable than fish-derived products. Limited fish stocks and expensive microbial culture processes encourage researchers to consider alternative plant-based production of these beneficial long-chain polyunsaturated fatty acids. There has already been considerable success in producing transgenic oilseed crops that synthesize fatty acids such as arachidonic acid, EPA, and DHA. Arachidonic acid itself has not been found in higher plants, but does occur in some algae, mosses, and ferns. All of the omega-3 fatty

acids are beneficial to health, but the plant and fish oils provide different compounds, and the distinction between the two groups should be recognized. Although most plant-derived oils contain high amounts of unsaturated fatty acid glycerides, including those of linoleic and α-linolenic acids, the conversion of linoleic acid into γ-linolenic acid can be blocked or inhibited in certain conditions in humans. This restricts synthesis of prostaglandins. In such cases, the use of food supplements, e.g. evening primrose oil from Oenothera biennis (Onagraceae) or borage oil from Borago officinalis (Boraginaceae), which are rich in γ-linolenic esters (see Table 3.1), can be valuable and help in the disorder. These plants are somewhat unusual in their ability to desaturate linoleic esters towards the carboxyl terminus via a 6 -desaturase, rather than towards the methyl terminus as is more common in plants. Expression of 6 -desaturase genes, either from plants or suitable fungi, can be used

O OPL

PL = phospholipid

oleic acid 18:1 (9c) O2

E1

O OPL

linoleic acid 18:2 (9c,12c) O2

E1

H

O OPL

crepenynic acid 18:2 (9c,12a) O2

H allylic isomerization

O OPL

dehydrocrepenynic acid 18:3 (9c,12a,14c) O

O OPL

OPL

18:3 (9c,12a,14a)

18:4 (9c,12a,14a,17) − CO2

O OPL

OH

18:4 (9c,12a,14a,16a) β-oxidations allylic isomerization CO2H dehydromatricaria acid 10:4 (2t,4a,6a,8a)

Figure 3.11

51

falcarinol E1: Δ12-desaturase/Δ12-acetylenase (bifunctional)

52 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition to increase the production of γ-linolenic and stearidonic acids in hosts such as soybeans. In the vast majority of unsaturated fatty acids, the double bonds have the Z/cis configuration. Fatty acids with trans double bonds do occur naturally at relatively low levels in meat and dairy products as a by-product of fermentation in ruminant animals. However, trans-unsaturation may be introduced during the partial hydrogenation of polyunsaturated fats that is commonly practised during food processing to produce semi-solid fats from oils. There now seems to be good correlation between the consumption of trans-fats and the occurrence of coronary heart disease and atherosclerosis, though precise mechanisms are yet to be determined. Nevertheless, levels of trans-fats in foodstuffs are now closely monitored. Many unsaturated compounds found in nature contain one or more acetylenic bonds, and these are predominantly produced by further desaturation of olefinic systems in fatty acid-derived molecules. They are surprisingly widespread in nature and are found in many organisms, but they are especially common in plants of the Compositae/Asteraceae, the Umbelliferae/Apiaceae, and fungi of the group Basidiomycetes. These compounds tend to be highly unstable and some are even explosive if sufficient amounts are accumulated. Since only very small amounts are present in plants, this does not present any widespread hazard. Whilst fatty acids containing several double bonds usually have these in a non-conjugated array, molecules containing triple bonds tend to possess conjugated unsaturation. This gives the compounds intense and highly characteristic UV spectra, which aids their detection and isolation. The processes of desaturation are exemplified in Figure 3.11, in which oleic acid (probably bound as a phospholipid) features as a precursor of crepenynic acid and dehydrocrepenynic acid. A bifunctional desaturase/acetylenase system catalysing these two steps has been characterized in the plant Crepis alpina (Compositae/Asteraceae). The acetylenic bond is now indicated by ‘a’ in the semi-systematic shorthand nomenclature. Chain shortening by β-oxidation (see page 18) is often a feature of these pathways, and formation of the C10 acetylenic acid dehydromatricaria acid proceeds through C18 intermediates, losing eight carbon atoms, presumably via four β-oxidations. In the latter part of the pathway, the Z double bond from oleic acid moves into conjugation with the polyacetylene chain via an allylic isomerization, giving the more favoured E-configuration. Loss of the carboxyl function is also encountered, and falcarinol is an example of such structures, most likely derived via dehydrocrepenynic

acid. Some noteworthy acetylenic structures (though they are no longer acids and components of fats) are given in Figure 3.12. Cicutoxin from the water hemlock (Cicuta virosa; Umbelliferae/Apiaceae) and oenanthotoxin from the hemlock water dropwort (Oenanthe crocata; Umbelliferae/Apiaceae) are extremely toxic to mammals, causing persistent vomiting and convulsions, leading to respiratory paralysis. Ingestion of the roots of these plants may frequently lead to fatal poisoning. Falcarinol is a constituent of Falcaria vulgaris (Umbelliferae/Apiaceae), Oenanthe crocata, Hedera helix (Araliaceae), and several other plants, and is known to cause contact dermatitis in certain individuals when the plants are handled. Falcarinol (sometimes called panaxynol) and the structurally related panaxytriol are also characteristic polyacetylene components of ginseng (Panax ginseng; Araliaceae; see page 245). Wyerone from the broad bean (Vicia faba; Leguminosae/Fabaceae) has antifungal properties, and its structure exemplifies how the original straight chain may be cross-linked to produce a ring system. The furan ring is believed to originate from a conjugated diyne. Primary amides of unsaturated fatty acids have been characterized in humans and other mammals, and although their biological role is not fully understood, they may represent a group of important signalling molecules.

OH

OH cicutoxin

OH

OH

oenanthotoxin

falcarinol (panaxynol)

OH

OH

Figure 3.12

OH panaxytriol

OH

O O wyerone

CO2Me

The Acetate Pathway: Fatty Acids and Polyketides Oleamide, the simple amide of oleic acid, has been shown to be a sleep-inducing lipid, and the amide of erucic acid, erucamide, stimulates the growth of blood vessels. The ethanolamide of arachidonic acid, anandamide, appears to be the natural ligand for receptors to which cannabinoids bind; this and related structures will be considered later (see page 122). The herbal preparation echinacea [Box 3.1] is derived from the roots

53

of Echinacea purpurea (Compositae/Asteraceae) and is used for its immunostimulant properties, particularly as a prophylactic and treatment for the common cold. At least some of its activity arises from a series of alkamides (also termed alkylamides), amides of polyunsaturated acids with isobutylamine. These acids are predominantly C11 and C12 diene-diynes (Figure 3.13).

Box 3.1 Echinacea Echinacea consists of the dried roots of Echinacea purpurea, Echinacea angustifolia, or Echinacea pallida (Compositae/ Asteraceae), herbaceous perennial plants indigenous to North America, and widely cultivated for their large daisy-like flowers, which are usually purple or pink. Herbal preparations containing the dried root, or extracts derived from it, are hugely popular, being promoted as immunostimulants, particularly as prophylactics and treatments for bacterial and viral infections, e.g. the common cold. Tests have validated stimulation of the immune response, though the origins of this activity cannot be ascribed to any specific substance. Activity has variously been assigned to lipophilic alkamides, polar caffeic acid derivatives, high molecular weight polysaccharide material, or to a combination of these. Compounds in each group have been demonstrated to possess some pertinent activity, e.g. immunostimulatory, anti-inflammatory, antibacterial, or antiviral effects. The alkamides comprise a complex mixture of unsaturated fatty acids as amides with 2-methylpropanamine (isobutylamine) or 2-methylbutanamine, amines which are probably decarboxylation products from valine and isoleucine respectively. The acid portions are predominantly C11 and C12 diene-diynes or tetraenes (Figure 3.13). These compounds are found throughout the plant, though relative proportions of individual components vary considerably. The root of Echinacea purpurea contains at least 12 alkamides (about 0.6%), of which C12 diene-diynes predominate; levels of these compounds fall significantly during drying and storage. Caffeic acid derivatives present include caffeic acid (see page 149), chlorogenic acid (5-O-caffeoylquinic acid, see page 150), caftaric acid (2-O-caffeoyltartaric acid), and cichoric acid (2,3-di-O-caffeoyltartaric acid) (Figure 3.13). Cichoric acid is a major component (0.6–2.1%) in Echinacea purpurea, but only minor in the other species. O

O Z

E

E

R

N H

N H

R = H or Me E

O

Z

O

N H

Z

R

N H

tetraene alkamides

R = H or Me O

O Z

E N H

diene-diyne alkamides

HO HO

OH

CO2H O

O HO2C

OH O

cichoric acid

Figure 3.13

UNCOMMON FATTY ACIDS Most fatty acids are undoubtedly primary metabolites. However, a considerable amount of structural diversity is encountered in this group of compounds, with some structures having a rather limited natural distribution, so

it is more appropriate to think of these as secondary metabolites. The distinction is actually unnecessary, since it is impossible to consider one group without the other; this is a typical ‘grey’ area. Some structures arise by further modification of the basic straight-chain systems

54 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O

PL = phospholipid

12

OPL oleic acid OH

E1

ricinoleic acid

O2/NADPH

pyrolysis

O

CO2H

OPL ricinoleic acid E1: oleate

undecenoic acid

Δ12-hydroxylase

Figure 3.14

already discussed, whilst others require a fundamental change in the nature of the starter and extender units employed in the biosynthesis. Ricinoleic acid (Figure 3.14) is the 12-hydroxy derivative of oleic acid and is the major fatty acid found in castor oil, expressed from seeds of the castor oil plant (Ricinus communis; Euphorbiaceae). It is formed by direct hydroxylation of oleic acid, esterified as part of a phospholipid, by the action of an O2 - and NADPH-dependent mixed-function oxidase. This is not of the cytochrome P-450 type, but structurally and mechanistically resembles the fatty acid desaturase enzymes. Castor oil has a long history of use as a domestic purgative, but it is now mainly employed as a cream base. Undecenoic acid (9 -undecenoic acid) can be obtained from ricinoleic acid by thermal degradation, and as the zinc salt or in ester form, it is used in a number of fungistatic preparations. Fatty acid hydroxylases of the cytochrome P-450 type have also been identified; the position of hydroxylation can vary according to enzyme, though hydroxylation of the terminal methyl (ω-hydroxylation) is quite common. Epoxy fatty acids like vernolic acid (Figure 3.15) have been found in substantial quantities in the seed oil of some plant species, including Vernonia galamensis and Stokesia laevis (both Compositae/Asteraceae). Vernolic O OPL linoleic acid E1

O

O2/NADPH

O OPL

vernolic acid E1: epoxygenase

Figure 3.15

PL = phospholipid

acid is formed by direct epoxidation of linoleic acid (as a phospholipid ester), but, as with hydroxylation, different types of enzyme have been identified. These plants also exploit desaturase-like systems, whereas the same transformation in Euphorbia lagascae (Euphorbiaceae) is catalysed by a cytochrome P-450-dependent enzyme. Whilst straight-chain fatty acids are the most common, branched-chain acids have been found to occur in mammalian systems, e.g. in wool fat and butter fat. They are also characteristic fatty acid constituents of the lipid part of cell walls in some pathogenic bacteria. Several mechanisms appear to operate in their formation. Methyl side-chains can be introduced by a C-alkylation mechanism using SAM. Tuberculostearic acid (Figure 3.16) is a C-methyl derivative of stearic acid found in the cell wall of Mycobacterium tuberculosis, the bacterium causing tuberculosis, and it provides a diagnostic marker for the disease. It is derived from oleic acid by alkylation on C-10, initiated by the double-bond electrons. Methyl transfer is followed by a Wagner–Meerwein 1,2-hydride shift in the carbocation, then proton loss to give the 10-methylene derivative. Finally, the double bond is reduced in an NADPH-dependent reaction to give tuberculostearic acid. This sequence of transformations is also seen during C-methylation of sterol side-chains (see page 252). Alternatively, loss of a proton from the first-formed carbocation intermediate via cyclopropane ring formation leads to dihydrosterculic acid. This is known to be dehydrogenated to sterculic acid, an unusual fatty acid containing a highly strained cyclopropene ring. Sterculic acid is present in the seed oil from Sterculia foetida (Sterculiaceae), and with similar cyclopropene acids, e.g. malvalic acid, is present in edible cottonseed oil from Gossypium species (Malvaceae). Malvalic acid is produced from sterculic acid by chain shortening from the carboxyl end (Figure 3.16). Sterculic acid is an inhibitor of the 9 -desaturase which converts stearic acid into oleic

The Acetate Pathway: Fatty Acids and Polyketides

55

Ad

electrophilic addition: SAM as alkylating agent

PL = phospholipid

SAM

H 3C S

O

R 10

OPL oleic acid E1

cation discharged by formation of cyclopropane ring and loss of proton O

E2

Wagner−Meerwein 1,2-hydride shift H3C

H O

H 2C

≡

OPL

H carbocation intermediate

OPL carbocation intermediate E2 O

H

OPL

OPL E1

dihydrosterculic acid

O

− H+

desaturation O

OPL 10-methylenestearic acid reduction of double bond

OPL

O

sterculic acid

NADPH

α-oxidation OPL

tuberculostearic acid

OPL

O

malvalic acid

O

E1: 10-methylenestearate synthetase E2: cyclopropane fatty acid synthase

Figure 3.16

SCoA O propionyl-CoA

E1

E1: propionyl-CoA carboxylase

CO2H

CO2, ATP biotin

SCoA

SCoA starter unit

extender unit

O methylmalonyl-CoA CO2H

O 2,4,6,8-tetramethyldecanoic acid

Figure 3.17 acid and is potentially harmful to humans in that it can alter membrane permeability and inhibit reproduction. Seed oils containing cyclopropene fatty acids destined for human consumption require suitable treatment to remove these undesirables. Methyl side-chains can also be introduced by using methylmalonyl-CoA instead of malonyl-CoA as the chain

extending agent (Figure 3.17). Methylmalonyl-CoA arises by biotin-dependent carboxylation of propionyl-CoA in exactly the same way as malonyl-CoA was formed (see page 17). 2,4,6,8-Tetramethyldecanoic acid found in the preen gland wax of the goose (Anser anser) is produced from an acetyl-CoA starter and four methylmalonyl-CoA chain extender units. This introduces the concept of

56 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition changing the starter and/or extender units whilst retaining the same FAS type of mechanism. It is a concept that gets developed much further with macrolide antibiotic structures (see page 68). Various bacteria contain a range of iso-fatty acids that help to control membrane fluidity. These iso-fatty acids are produced by malonate chain extension of a number of different starter units derived from branched-chain amino acids, modified into CoA-esters. Thus, leucine, valine, and isoleucine may generate isovaleryl-CoA, isobutyryl-CoA, and 2-methylbutyryl-CoA respectively as starter groups (Figure 3.18). Several other examples where these amino acids provide building blocks will be met in due course. NH2

Chaulmoogric and hydnocarpic acids (Figure 3.19) are uncommon cyclopentenyl fatty acids found in chaulmoogra oil, an oil expressed from seeds of Hydnocarpus wightiana (Flacourtiaceae). These acids are known to arise by malonate chain extension of the coenzyme A ester of 2-cyclopentenyl carboxylic acid as an alternative starter unit to acetate. Chaulmoogra oil provided for many years the only treatment for the relief of leprosy, these two acids being strongly bactericidal towards the leprosy infective agent Mycobacterium leprae. Purified salts and esters of hydnocarpic and chaulmoogric acids were subsequently employed, until they were then themselves replaced by more effective synthetic agents. NH2

CO2H

NH2

CO2H

leucine

CO2H valine

isoleucine

E1

E1

E1

E2

E2

E2 O

O

O SCoA

SCoA isovaleryl-CoA

2-methylbutyryl-CoA

SCoA isobutyryl-CoA

malonyl-CoA

malonyl-CoA

malonyl-CoA

E3

E3

E3 CO2H

CO2H 12-methyltridecanoic acid

15-methylhexadecanoic acid E1: transaminase E2: branched-chain keto acid dehydrogenase E3: fatty acid synthase

CO2H 12-methyltetradecanoic acid

Figure 3.18

SCoA 2-cyclopentenylcarboxyl-CoA

Figure 3.19

CO2H

malonyl-CoA

O

hydnocarpic acid CO2H chaulmoogric acid

The Acetate Pathway: Fatty Acids and Polyketides O SCoA linoleic acid

intermediates are shown as CoA esters, though this has not been confirmed

CoAS

b-oxidation

O

O HO2C hexylmalonyl thioester

SCoA

tetradeca-5,8-dienoic acid hydroxylation OH

CoAS

O

O

alternatively nucleophile may be malonyl thioester

SCoA

octanoic acid

Claisen reaction OH

O

COSCoA

reduction OH

OH

COSCoA

lactone formation

O OH

CO2H

O

COSCoA

NH2 Leu

esterification followed by N-formylation

NH2

H N

O

O

O

O O

lipstatin CO2H

CoAS

NH2 Leu

H N

O

O

O

O O

O

barnched-chain lipstatin analogue

Figure 3.20

HO2C NH2 Leu

57

58 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Some branched-chain systems can be rationalized as a combination of two separate fatty acid chains coupled in a Claisen reaction. This has been shown for the β-lactone derivative lipstatin found in Streptomyces toxytricini, a compound that generated considerable interest because of its ability to inhibit pancreatic lipase; it has since been developed into an anti-obesity drug [Box 3.2]. Lipstatin is formed from the two fatty acids tetradeca-5,8-dienoic acid and octanoic acid; the first of these is believed to originate from linoleic acid derived from sunflower oil in the culture medium (Figure 3.20). There is evidence that the nucleophilic species is more

likely to be the activated hexylmalonyl thioester rather than the octanoyl thioester. Claisen coupling is followed by simple reduction and lactonization (esterification). The remaining portion of the molecule is leucine derived, and esterification appears to precede introduction of the N-formyl group; the N-formyl group is presumably an oxidized N-methyl. A branched-chain analogue of lipstatin is a minor metabolite in the cultures (Figure 3.20). This incorporates two molecules of leucine, one of which acts as a starter unit for the methyloctanoate fatty acid portion, most likely via isovaleryl-CoA as seen above.

Box 3.2 Lipstatin Lipstatin was isolated from the mycelium of Streptomyces toxytricini cultures and shown to possess marked inhibitory activity towards pancreatic lipase, the key enzyme for intestinal fat digestion. The β-lactone function in the lipophilic molecule irreversibly inactivates lipase by covalent reaction with a serine residue at the catalytic site; the reaction closely parallels that of serine residues with β-lactam antibiotics (see page 464). Tetrahydrolipstatin (orlistat) (Figure 3.21), obtained by catalytic hydrogenation of lipstatin, was selected for further development; it is more stable and crystallizes readily, although it is somewhat less active. Orlistat is now manufactured by total synthesis.

H N

O

O

O

O O

orlistat (tetrahydrolipstatin)

Figure 3.21 Orlistat reduces the absorption of dietary fat and is used in conjunction with a low-fat calorie-reduced diet to reduce body mass in obese patients. Only trace amounts of the drug are absorbed systemically, and the primary effect is local lipase inhibition within the digestive tract; dietary fat is thus excreted undigested. Absorption of fat-soluble vitamins, especially vitamin D, is also inhibited, and vitamin supplements are usually co-administered.

PROSTAGLANDINS The prostaglandins are a group of modified C20 fatty acids first isolated from human semen and initially assumed to be secreted by the prostate gland [Box 3.3]. They are now known to occur widely in animal tissues, but only in tiny amounts, and they have been found to exert a wide variety of pharmacological effects on humans and animals. They are active at very low, hormone-like concentrations

and can regulate blood pressure, contractions of smooth muscle, gastric secretion, and platelet aggregation. Their potential for drug use is extremely high, but it has proved difficult to separate the various biological activities into individual agents. The basic prostaglandin skeleton is that of a cyclized C20 fatty acid containing a cyclopentane ring, a C7 side-chain with the carboxyl function, and a C8 side-chain with the methyl terminus. Prostaglandins

The Acetate Pathway: Fatty Acids and Polyketides 8

8

5

11

14

dihomo-γ-linolenic

11

(Δ8,11,14)

O

14

11

(Δ5,8,11,14)

arachidonic

OH PGE1

5

14

17

eicosapentaenoic (Δ5,8,11,14,17)

CO2H

CO2H

HO

CO2H

O

O CO2H

HO

8

CO2H

CO2H

59

OH PGE2

HO

OH PGE3

Figure 3.22 are biosynthesized from three EFAs, namely Δ8,11,14 eicosatrienoic acid (dihomo-γ-linolenic acid), Δ5,8,11,14 eicosatetraenoic acid (arachidonic acid), and Δ5,8,11,14,17 -eicosapentaenoic acid, which yield prostaglandins of the 1-, 2-, and 3-series respectively (Figure 3.22; see Figure 3.24 below for principles of nomenclature). The three precursors lead to products of similar structure, but with varying levels of unsaturation in the two side-chains. Some of the structures elaborated from arachidonic acid are shown in Figure 3.23. In the first reaction, arachidonic acid is converted into prostaglandin H2 (PGH2 ) by way of prostaglandin G2 (PGG2 ) by prostaglandin H synthase. This is a bifunctional enzyme comprised of an oxygenase (cyclooxygenase, COX) and a peroxidase, both requiring haem as cofactor. COX incorporates two molecules of oxygen, liberating a compound with both cyclic and acyclic peroxide functions, and also creating five chiral centres. In arachidonic acid, the methylene group flanked by two double bonds is susceptible to radical oxidation, initiated by a tyrosyl radical in the enzyme. This leads to incorporation of oxygen and formation of a cyclic peroxide function. Radical addition to an alkene function allows ring formation; then, a further incorporation of oxygen, in what is effectively a repeat of the earlier sequence, leads to an acyclic peroxide and produces PGG2 . The hydrogen atom terminating the synthesis is obtained from the same tyrosine function in the enzyme, thus propagating the radical reaction. The acyclic peroxide group in PGG2 is then cleaved by the peroxidase component of prostaglandin H synthase to yield PGH2 . This unstable peroxide occupies a central role and can be modified in several different ways. Reductive cleavage gives prostaglandin

F2α (PGF2α ); other modifications can be rationally accommodated by initial cleavage of the cyclic peroxide to the diradical, though alternative ionic mechanisms may also be proposed. Quenching of the radicals by capture and loss of hydrogen atoms would provide either prostaglandin E2 (PGE2 ) or prostaglandin D2 (PGD2 ). Reduction of PGE2 is known to provide a minor pathway to PGF2α . The bicyclic system in prostaglandin I2 (PGI2 ; prostacyclin) is envisaged as arising by involvement of a side-chain double bond, then loss of a hydrogen atom, all catalysed by prostacyclin synthase, a cytochrome P-450-dependent enzyme. Prostaglandin structures representative of the 1-series, e.g. PGE1 , or of the 3-series, e.g. PGE3 , can be formed in a similar way from the appropriate fatty acid precursor (Figure 3.22). The basic skeleton of the prostaglandins is termed prostanoic acid, and derivatives of this system are collectively known as prostanoids. The term eicosanoids is also used to encompass prostaglandins, thromboxanes, and leukotrienes, which are all derived from C20 fatty acids (eicosanoic acids). Semi-systematic nomenclature of prostaglandins is based on the substitution pattern in the five-membered ring, denoted by a letter suffix (Figure 3.24), and the number of double bonds in the side-chains is given by a numerical subscript. Greek letters α and β are used to indicate the configuration at C-9, α indicating that the substituent is below the plane (as found in natural prostaglandins) and β indicating that the substituent is above the plane (as in some synthetic analogues). ‘Prostaglandin’ is usually abbreviated to PG. Prostaglandins A, B, and C are inactive degradation products from natural prostaglandins.

60 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition methylene flanked by double bonds is susceptible to radical oxidation

resonance-stabilized radical

−H O2

CO2H H H

CO2H

CO2H

E1 arachidonic acid

O

O

radical addition to double bond; formation of cyclic peroxide

CO2H

radical addition to O2 and formation of peroxide radical

O

CO2H

O

O

O

radical addition to double bond; formation of cyclopentane

resonance-stabilized radical

O

O

CO2H

O

CO2H

O O O E1

O

reductive cleavage of acyclic peroxide

CO2H

E2

O

E3

radical addition to O2 and formation of peroxide radical; peroxide radical abstracts H atom CO2H

O cyclic peroxide

PGH2

OH

O

H

O OH acyclic peroxide

NADPH

PGG2

HO CO2H radical cleavage of cyclic peroxide

NADPH HO

O

CO2H

OH

PGF2a

O

E7

CO2H

HO

–H ,+H

OH

E5 HO

O OH

E4

E6

CO2H

O

O2 NADPH

OH

O O

H

O

CO2H E4

O OH E1: cyclooxygenase (COX) E2: peroxidase E1 + E2 = prostaglandin H synthase

Figure 3.23

PGD2

CO2H

CO2H

H

PGE2

HO

OH

E3: prostaglandin F synthase E4: prostaglandin I synthase (prostacyclin synthase)

HO

OH

E5: prostaglandin E synthase E6: prostaglandin D synthase E7: prostaglandin E2 9-reductase

PGI2

The Acetate Pathway: Fatty Acids and Polyketides O

O

R1

O

R1

R2

R1

R2

HO

R2

R1

PGB

PGC

HO

R1

R2 O

PGA

O

PGD

R1

R2

R2

HO

CO2H

HO PGE

61

PGF O

O

R1

O PGG

O OH

O

R1

O

R2 PGH

R2 HO

R1

9

8

7

5

6

4

3

12

13

14

15

16

17

prostanoic acid

R2

1-series:

CO2H

2-series:

CO2H

1 2

18

OH

CO2H

10 11

PGI

20

OH

19

3-series:

CO2H OH

Figure 3.24

Box 3.3 Prostaglandins Prostaglandins occur in nearly all mammalian tissues, but only at very low concentrations. PGE1 and PGF1α were initially isolated from sheep seminal plasma, but these compounds and PGD2 , PGE2 , and PGF2α are widely distributed. Animal sources cannot supply sufficient amounts for drug usage. The soft coral Plexaura homomalla (sea whip) from the Caribbean has been identified as having very high (2–3%) levels of prostaglandin esters, predominantly the methyl ester of PGA2 (1–2%) with related structures. Prostaglandins of the A-, E-, and F-types are widely distributed in soft corals, especially Plexaura, though many corals produce prostaglandin structures not found in animals. For several years, the sea whip coral was used as a source of research material, but this was neither a satisfactory nor renewable natural source. Considerable effort was exerted on the total synthesis of prostaglandins and their interconversions, and the high level of success achieved opened up the availability of compounds for pharmacological testing and subsequent drug use. Synthetic analogues have also been developed to modify or optimize biological activity. The studies have demonstrated that biological activity is effectively confined to the natural enantiomers; the unnatural enantiomer of PGE1 had only 0.1% of the activity of the natural isomer. The prostaglandins display a wide range of pharmacological activities, including contraction and relaxation of smooth muscle of the uterus, the cardiovascular system, the intestinal tract, and of bronchial tissue. They also inhibit gastric acid secretion, control blood pressure and suppress blood platelet aggregation, as well as acting as mediators of inflammation, fever, and allergy. Some of these effects are consistent with the prostaglandins acting as second messengers, modulating transmission of hormone stimulation and, thus, metabolic response. They are synthesized in response to various stimuli in a variety of cells, released immediately after synthesis, and act in the vicinity of their synthesis to maintain local homeostasis. Some prostaglandins in the A and J series have demonstrated potent antitumour properties. Since the prostaglandins control many important physiological processes in animal tissues, their drug potential is high, but the chances of precipitating unwanted side-effects are also high, and this has so far limited their therapeutic use. There is, however, much additional scope for controlling the production of natural prostaglandins in body tissues by means of specific inhibitors. Indeed, it has been found that some established non-steroidal anti-inflammatory drugs (NSAIDs), e.g. aspirin, indometacin, and

62 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.3 (continued) ibuprofen, inhibit early steps in the prostaglandin biosynthetic pathway that transform the unsaturated fatty acids into cyclic peroxides. The local production of prostaglandins such as PGE2 can sensitize pain nerve endings and increase blood flow, promoting pain, swelling, and redness. On the other hand, PGI2 and PGE2 are protective to the stomach, and inhibiting their formation explains the gastrointestinal toxicity associated with prolonged and high-dose use of NSAIDs. Aspirin is now known to inactivate irreversibly the COX activity (arachidonic acid → PGG2 ), though not the peroxidase activity (PGG2 → PGH2 ), by selective acetylation of a serine residue of the enzyme; ibuprofen and indometacin compete with arachidonic acid at the active site and are reversible inhibitors of COX. A more recent discovery is that two forms of the COX enzyme exist, designated COX-1 and COX-2. COX-1 is expressed constitutively in most tissues and cells and is thought to control synthesis of those prostaglandins important for normal cellular functions, such as gastrointestinal integrity and vascular homeostasis. In simplistic terms, COX-1 is considered a ‘housekeeping’ enzyme. COX-2 is not normally present, but is inducible in certain cells in response to inflammatory stimuli, resulting in enhanced prostaglandin release in the central nervous system and inflammatory cells with the characteristic inflammatory response. Its inhibition appears to produce the analgesic, antipyretic, and anti-inflammatory effects of NSAIDs. However, popular NSAIDs do not discriminate between the two COX enzymes, and so this leads to both therapeutic effects via inhibition of COX-2 and adverse effects such as gastrointestinal problems, ulcers, and bleeding via inhibition of COX-1. Because of differences in the nature of the active sites of the two enzymes, it has now been possible to develop agents that can inhibit COX-2 rather than COX-1 as potential new anti-inflammatory drugs. The first of these ‘coxib’ drugs, celecoxib and rofecoxib, were introduced for relief of pain and inflammation in osteoarthritis and rheumatoid arthritis, though rofecoxib was subsequently withdrawn because of significant cardiovascular side-effects. Second-generation agents etoricoxib and lumiracoxib have since been introduced. The widely used analgesic paracetamol (US: acetaminophen) is usually classified as an NSAID, though it has little anti-inflammatory activity. Its ability to inhibit pain and fever is suggested to emanate from inhibition of a COX-1 variant, termed COX-3. The anti-inflammatory activity of corticosteroids correlates with inhibition of phospholipase enzymes and preventing the release of arachidonic acid from storage phospholipids, but expression of COX-2 is also inhibited by glucocorticoids. The role of of essential fatty acids (see page 49) such as linoleic and γ-linolenic acids, obtained from plant ingredients in the diet, can now readily be appreciated. Without a source of arachidonic acid, or compounds which can be converted into arachidonic acid, synthesis of prostaglandins would be compromised, and this would seriously affect many normal metabolic processes. A steady supply of prostaglandin precursors is required; prostaglandins are continuously being synthesized and then degraded. Prostaglandins are rapidly degraded by processes which include oxidation of the 15-hydroxyl to a ketone, reduction of the 13,14-double bond, and oxidative degradation of both side-chains. A major area of application of prostaglandins as drugs is in obstetrics, where they are used to induce abortions during the early to middle stages of pregnancy, or to induce labour at term. Dinoprost (PGF2α ) is not usually prescribed, since it is rapidly metabolized in body tissues (half-life less than 10 min), and the modified version carboprost (15-methyl PGF2α ; Figure 3.25) has been developed to reduce deactivation by blocking oxidation at position 15. Carboprost is produced by oxidizing the 15-hydroxyl in a suitably protected PGF2α , then alkylating the 15-carbonyl with a Grignard reagent. Carboprost is effective at much reduced dosage than dinoprost and is of value in augmenting labour at term, especially in cases where ergometrine (see page 394) or oxytocin (see page 430) are ineffective. The natural prostaglandin structure dinoprostone (PGE2 ) is also used primarily to induce labour; the 16-dimethyl prostaglandin gemeprost is currently the preferred prostaglandin for abortions. These agents are usually administered vaginally. Alprostadil (PGE1 ) differs from PGE2 by having unsaturation in only one side-chain. Though having effects on uterine muscle, it also has vasodilator properties, and these are exploited for maintaining new-born infants with congenital heart defects, facilitating blood oxygenation prior to corrective surgery. The very rapid metabolism of PGE1 means this drug must be delivered by continuous intravenous infusion. Alprostadil is also of value in male impotence, self-injectable preparations being used to achieve erection of the penis. An interesting modification to the structure of PGE1 is found in the analogue misoprostol. This compound has had the oxygenation removed from position 15, transferred to position 16, plus alkylation at position 16 to reduce metabolism (compare 15-methyl PGF2α above). These modifications result in an orally active drug which inhibits gastric secretion effectively and can be used to promote healing of gastric and duodenal ulcers. In combination with non-specific NSAIDs, it can significantly lower the incidence of gastrointestinal side-effects such as ulceration and bleeding. It also induces labour, and is occasionally used for this purpose. Epoprostenol (PGI2 , prostacyclin) reduces blood pressure and also inhibits platelet aggregation by reducing calcium concentrations. It is employed to inhibit blood clotting during renal dialysis, but its very low half-life (about 3 min) again necessitates continuous intravenous administration. The tetrahydrofuran ring is part of an enol ether and is readily opened by hydration, leading to 6-ketoprostaglandin F1α (Figure 3.26). Iloprost (Figure 3.25) is a stable carbocyclic analogue of potential use in the treatment of thrombotic diseases.

The Acetate Pathway: Fatty Acids and Polyketides

63

Box 3.3 (continued) HO

O

O CO2H

CO2Me

CO2H

15

Me

HO

16

OH

HO

HO

OH dinoprostone (PGE2)

carboprost (15-methyl PGF2a ) O

O

CO2H

CO2H

OH gemeprost

HO

O O

HO

HO

OH alprostadil (PGE1) CO2H

RS HO 16 Me misoprostol

HO

HO R

R = H, latanoprost R = CF3, travoprost

CO2H

HO

NHEt

O

O RS

HO

HO

OH

HO

HO

OH

epoprostenol (PGI2/prostacyclin)

bimatoprost

iloprost

Figure 3.25

CO2H H

hydrolysis of enol ether

O

hemiketal formation

H 2O O

HO H

O

hydrolysis of hemiketal O HO

CO2H 6

H+

HO

OH PGI2

HO

HO

HO

OH 6-keto PGF1a

Figure 3.26

Latanoprost, travoprost, and bimatoprost (Figure 3.25) are recently introduced prostaglandin analogues which increase the outflow of aqueous humour from the eye. They are thus used to reduce intraocular pressure in the treatment of the eye disease glaucoma. Bimatoprost is an amide derivative related to prostaglandin amides (prostamides) obtained by the action of COX-2 on anandamide, the natural ligand for cannabinoid receptors (see page 122). The pharmacological properties of prostamides cannot readily be explained simply by interaction with prostaglandin receptors; bimatoprost also inhibits prostaglandin F synthase. An unusual side-effect of bimatoprost is also being exploited cosmetically: it makes eyelashes grow longer, thicker, and darker.

64 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.3 (continued) Isoprostanes Isoprostanes represent a new class of prostaglandin-like compounds produced in vivo in humans and animals by non-enzymic radical-mediated oxidation of membrane-bound polyunsaturated fatty acids independent of the COX enzyme. An isomer of PGF2α in which the two alkyl substituents on the five-membered ring were arranged cis rather than trans was detected in human urine and was the first of these compounds to be characterized. This compound was initially termed 8-iso-PGF2α , or 8-epi-PGF2α ; however, as many more variants in the isoprostane series were discovered, it is now termed 15-F2t -IsoP (Figure 3.27). The first number refers to the position of the hydroxyl, F2 relates the compound to the prostaglandin class, and then t (trans) or c (cis) defines the relationship of the side-chain to the ring hydroxyls. The isoprostanes can be viewed as arising by a radical mechanism which resembles the enzyme-mediated formation of prostaglandins shown in Figure 3.23. Varying side-chain substituents arise by utilizing different double bonds from the several available in the cyclization mechanism and incorporating an oxygen atom from molecular oxygen at different positions. Many variants are formed because chemical processes rather than enzyme-controlled processes are employed. Isoprostanes derived similarly from docosahexaeneoic acid (DHA), a major lipid component of brain tissue, have also been isolated, and these are termed neuroprostanes. Structurally related compounds are also found in plants; these are derived from α-linolenic acid and are termed phytoprostanes (see page 204). Interest in these isoprostanoid derivatives stems partly from the finding that certain compounds possess biological activity, probably via interaction with receptors for prostaglandins. For example, 15-F2t -IsoP is a potent vasoconstrictor and also aggregates platelets. There is also evidence that prostaglandins, including PGE2 , PGD2 , and PGF2α , can also be formed via epimerization reactions on isoprostanes, i.e. prostaglandin synthesis by a chemical pathway independent of COX. Perhaps the most promising application relates to their origin via radical peroxidation of unsaturated fatty acids. Radicals are implicated in inflammatory and degenerative diseases such as atherosclerosis, cancer, and Alzheimer’s disease. Isoprostane analysis of urine or serum provides non-invasive monitoring of oxidative damage as an insight into these disease states.

HO

HO 8

12

HO

CO2H

CO2H

15

OH PGF2a

HO

OH 15-F2t-IsoP

Figure 3.27

THROMBOXANES

LEUKOTRIENES

An intriguing side-branch from the prostaglandin pathway leads to thromboxanes (Figure 3.28) [Box 3.4]. The peroxide and cyclopentane ring functions of PGH2 are cleaved and restructured to form thromboxane A2 (TXA2 ), which contains a highly strained four-membered oxetane ring. A hypothetical radical mechanism is shown; the enzyme also produces equimolar amounts of hydroxyheptadecatrienoic acid and malondialdehyde by a fragmentation reaction. TXA2 is highly unstable and reacts readily with nucleophiles. In an aqueous environment, it reacts to yield the biologically inactive hemiacetal thromboxane B2 (TXB2 ).

Yet another variant for the metabolism of arachidonic acid is the formation of leukotrienes, a series of fatty acid derivatives with a conjugated triene functionality, and first isolated from leukocytes [Box 3.5]. In a representative pathway (others have been characterized) (Figure 3.29), arachidonic acid is converted into a hydroperoxide, the point of oxygenation being C-5, rather than C-11 as in the prostaglandin pathway (Figure 3.23). This compound loses water via formation of an epoxide ring, giving leukotriene A4 (LTA4 ). This unstable allylic epoxide may hydrolyse by conjugate addition to give leukotriene B4 (LTB4 ), or alternatively the epoxide may be

The Acetate Pathway: Fatty Acids and Polyketides O

O

O

CO2H O

OH

O

OH

PGH2

HO CO2H

O acetal

OH TXA2

O CO2H

O O

O

malondialdehyde

O

hemiacetal

CO2H

O

OH

≡

E1

highly strained acetal ring system is susceptible to nucleophilic attack H2O H2O

HO

CO2H

CO2H E1

O

65

OH E1 CO2H

OH TXB2

E1: thromboxane A2 synthase

OH 12-hydroxy-5,8,10-heptadecatrienoic acid

Figure 3.28

Box 3.4 Thromboxanes The thromboxanes were isolated from blood platelets, and whilst TXA2 showed high biological activity, TXB2 was effectively inactive. TXA2 is a potent stimulator of vasoconstriction and platelet aggregation. Aggregation of blood platelets to form a clot or thrombus is caused by increasing cytoplasmic calcium concentrations and thus deforming the platelets, which then fuse together. TXA2 has the opposite effect to PGI2 , and presumably the development of thrombosis reflects an imbalance in the two activities. Both compounds are produced from the same precursor, PGH2 , which is converted in the blood platelets into TXA2 , and in the blood vessel wall into PGI2 . Thromboxanes A3 and B3 have also been isolated from blood platelets. These are derived from 5,8,11,14,17 -eicosapentaenoic acid and relate structurally to prostaglandins in the 3-series. TXA3 is not strongly aggregatory towards blood platelets. The highly unstable nature of the biologically active thromboxanes has made their synthesis difficult, and drug use of natural structures will probably be impracticable. It is likely that most efforts will be directed towards thromboxane antagonists to help reduce blood platelet aggregation in thrombosis patients. The value of aspirin in preventing cardiovascular disease is now known to be related to inhibition of thromboxane A2 biosynthesis in platelets.

Box 3.5 Leukotrienes The leukotrienes are involved in allergic responses and inflammatory processes. An antigen–antibody reaction can result in the release of compounds such as histamine (see page 398) or materials termed slow reacting substance of anaphylaxis (SRSA). These substances are then mediators of hypersensitive reactions such as hay fever and asthma. Structural studies have identified SRSA as a mixture of LTC4 , LTD4 , and LTE4 . These cysteine-containing leukotrienes are powerful bronchoconstrictors and vasoconstrictors, and induce mucus secretion, the typical symptoms of asthma. LTE4 is some 10–100-fold less active than LTD4 , so that degradation of the peptide side-chain represents a means of eliminating leukotriene function. LTB4 appears to facilitate migration of leukocytes in inflammation, and is implicated in the pathology of psoriasis, inflammatory bowel disease, and arthritis. The biological effects of leukotrienes are being actively researched to define the cellular processes involved. This may lead to the development of agents to control allergic and inflammatory reactions. Drugs inhibiting the formation of LTC4 and LTB4 are in clinical trials, whilst montelukast and zafirlukast have been introduced as orally active leukotriene (LTD4 ) receptor antagonists for the prophylaxis of asthma.

66 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition this reaction is analogous to that initiating prostaglandin formation (Figure 3.23)

O O

O OH

CO2H

CO2H H H

formation of acyclic peroxide

H

CO2H H

E1

hydroperoxide

arachidonic acid

conjugate nucleophilic attack of H2O onto epoxide H 2O

HO

cleavage of peroxide allows formation of epoxide; overall a dehydration reaction

E1

O

CO2H

CO2H

OH

E2

LTB4

LTA4 nucleophilic attack of thiol onto epoxide

glutathione (γ-glutamyl-cysteinyl-glycine)

E3

OH

OH CO2H

E4

S

O H N

H2N LTD4

CO2H

O – Gly

CO2H

– Glu

partial hydrolysis of glutathione portion

HO2C

H N

N H

NH2 LTC4

E5

S

Glu

CO2H

O Cys

Gly

OH CO2H S

LTE4

H2N

CO2H

E1: E2: E3: E4: E5:

5-lipoxygenase LTA4 hydrolase LTC4 synthase γ-glutamyltranspeptidase dipeptidase

Figure 3.29

attacked directly by a nucleophile, in this case the sulfur atom of the tripeptide glutathione (γ-glutamylcysteinyl-glycine; Figure 3.29). The adduct produced in the latter reaction is termed leukotriene C4 (LTC4 ). Partial hydrolysis in the tripeptide fragment then leads to leukotriene D4 (LTD4 ) and leukotriene E4 (LTE4 ). Analogues, e.g. LTA3 and LTA5 , are also known, and these are derived from Δ5,8,11 -eicosatrienoic acid and Δ5,8,11,14,17 -eicosapentaenoic acid respectively. The subscript numeral indicates the total number of double bonds in the leukotriene chain.

POLYKETIDE SYNTHASES: GENERALITIES During fatty acid biosynthesis, the growing chain is constructed by a Claisen reaction, catalysed by the ketosynthase activity in the FAS complex. This reaction initially produces a β-ketoester, and the ketone group is reduced after each condensation step and before the next round of chain extension (Figure 3.2). The overall reduction is achieved by a three-stage process: reduction to an alcohol (KR), dehydration to the conjugated ester (DH), then reduction of the double bond (ER). These reactions

The Acetate Pathway: Fatty Acids and Polyketides O

O

HO2C

SCoA malonyl-CoA

load extender unit

MAT

SACP

RCH2

KR: ketoreductase KS: ketosynthase MAT: malonyl/acetyl transferase

transfer acyl group; repeat cycle

O

O HO2C

ACP: acyl carrier protein AT: acetyl transferase DH: dehydratase ER: enoyl reductase

CH3 SCoA acetyl-CoA load AT starter unit

67

SEnz Enz−SH

Claisen KS reaction

O RCH2

KR

O SACP

β-ketoester

RCH2

reduction

DH

H OH O

O

O

O

varying amounts of reduction/dehydration

all keto groups reduced

O

O OH

RCH2

O

SACP RCH2 SACP RCH2 SACP hydroxyester dehydration conjugated ester reduction reduced ester

no reduction of keto groups O

ER

O

O

O

SACP RCH2 aromatics

SACP

macrolides

RCH2

SACP

fatty acids

Figure 3.30

are shown again in Figure 3.30. This figure also shows how the same general processes may be modified to produce other polyketide systems, namely aromatics and macrolides; these processes are catalysed by polyketide synthases (PKSs). For aromatics, the reduction sequence is omitted completely (or almost completely; see page 101) so that the β-ketoester re-enters the extension cycle, giving a poly-β-ketoester with alternate keto and methylene functions. This highly reactive poly-β-keto backbone undergoes further enzyme-catalysed intramolecular cyclization reactions which are responsible for generating a range of aromatic structures (see page 99). Macrolides are characterized by having carbon chains that have undergone partial, complete, or no reduction, according to the position in the chain. The chain may contain a mixture of hydroxyl groups, carbonyl groups, double bonds, and methylene groups. This means that the enzyme activities KR, DH, and ER are not all active during a particular extension cycle, and that a partially modified system, a β-ketoester, hydroxy ester, conjugated ester, or reduced ester, feeds back into the extension cycle. The order in which the modifications occur (or do not occur) is closely controlled by the enzyme. A detailed study of the genes, protein amino acid sequences, and mechanistic similarities in various PKS

enzymes has led to three general types being distinguished. As with the type I and type II FASs (see page 40), type I PKSs are very large multifunctional proteins with individual functional domains, whilst type II PKSs are composed of a complex of individual monofunctional proteins. Type I systems can also be subdivided into ‘iterative’ (i.e. repeating) and ‘noniterative’ categories. Iterative systems (like the FASs) use their functional domains repeatedly to produce a particular polyketide. Non-iterative systems possess a distinct active site for every enzyme-catalysed step. Type II systems are of the iterative type. Both types of system use ACP to activate acyl-CoA substrates and to channel the growing polyketide intermediates. Type I enzymes are responsible for macrolide biosynthesis, where the variability at each step in the biosynthetic pathway gives rise to much more structural diversity than encountered with fatty acids. The usual starter unit employed is either acetyl-CoA or propionyl-CoA, whilst malonyl-CoA or methylmalonyl-CoA is the main extender unit. Aromatic compounds are usually products from type II enzymes; though variation in starter units may be encountered, the chain extender unit is always malonyl-CoA. PKSs responsible for chain extension of cinnamoyl-CoA starter units leading to flavonoids and stilbenes (see page 116)

68 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition do not fall into either of the above categories. These enzymes differ from the other types in that they are homodimeric proteins, they utilize coenzyme A esters rather than ACPs, and they employ a single active site to perform a series of decarboxylation, condensation, cyclization, and aromatization reactions. They have now been termed type III PKSs or chalcone synthase-like PKSs, though many examples outside of the flavonoid group are now recognized. Type I PKSs are found in bacteria and fungi, type II PKSs are restricted to bacteria, whilst type III PKSs are found in plants, bacteria, and fungi. Despite the differences in molecular architecture, the chemical aspects of chain construction are effectively the same in all PKSs. In all cases, these natural products are produced by Claisen reactions involving thioesters, and a basic chain that is composed of a linear sequence of C2 units is constructed. Even when the starter unit is modified, or methylmalonyl-CoA is used as an extender unit instead of malonyl-CoA, the fundamental chain is effectively the same and is produced in the same mechanistic

manner. Therefore, notwithstanding the various building blocks employed, these compounds are all still considered as derived via the acetate pathway. It is also more fitting to discuss the compounds in groups according to structural aspects rather than the protein type involved in their construction. POLYKETIDE SYNTHASES: MACROLIDES The macrolides are a large family of compounds, many with antibiotic activity, characterized by a macrocyclic lactone (sometimes lactam) ring. Rings are commonly 12-, 14-, or 16-membered, though polyene macrolides (see page 81) are larger in the range 26–38 atoms. The largest natural macrolide structure discovered has a 66-membered ring. The macrolide antibiotics provide us with excellent examples of natural products conforming to the acetate pathway, but composed principally of propionate units, or mixtures of propionate and acetate units. There is now extensive genetic evidence from a variety of systems to show that macrolide assembly is O

O starter unit

extender units

6-hydroxylation

CO2H SCoA

OH

SCoA

+ 6×

O

E1

O

O O

OH

O2 NADPH

6

OH

OH

E2

O O

OH

6-deoxyerythronolide B

HO 11 10

9

E3

O 1

O

OH

7

OH

12 13

HO O

O

O

OMe

O

erythromycin A

OH

NMe2 HO

O

O

O

E4 O

O

OH

O-methylation of mycarose O

L-cladinose

E1: EryA (6-deoxyerythronolide B synthase; DEBS) E2: EryF (6-deoxyerythronolide B hydroxylase) E3: EryBV (mycarosyl transferase)

Figure 3.31

TDP-D-desosamine

OH

erythromycin C E5

TDP-L-mycarose

OH

12

O2 NADPH

SAM

3

erythronolide A

12-hydroxylation

D-desosamine NMe2

6 5 4

2

E4

O 8

OH

erythronolide B

2× glycosylation O

OH

D-desosamine

OH L-mycarose

erythromycin D E4: EryCIII (desosaminyl transferase) E5: EryK (erythromycin 12-hydroxylase) E6: EryG (methyltransferase)

The Acetate Pathway: Fatty Acids and Polyketides most often accomplished by non-iterative type I PKSs. Conceptually, polyketide synthesis by this group of enzymes is probably the easiest to envisage and understand, and provides the best grounding for appreciation of other systems. The PKS provides a biological production line of multifunctional proteins organized as discrete modules. Each module contains the enzyme activities for an extension cycle with its subsequent modifications. The developing polyketide chain attached to an ACP is thus modified according to the appropriate enzyme activities present, and is then passed on to another ACP prior to the next condensation and modification. The linear sequence of modules in the enzyme corresponds to the generated sequence of extender units in the polyketide product. Erythromycin A (Figure 3.31) from Saccharopolyspora erythraea is a valuable antibacterial drug [Box 3.6] and contains a 14-membered macrocycle composed entirely of propionate units, both as starter and extension units, the latter via methylmalonyl-CoA. In common with many antibacterial macrolides, sugar

69

units, including amino sugars, are attached through glycoside linkages. These unusual 6-deoxy sugars are frequently restricted to this group of natural products. In erythromycin A, the sugars are l-cladinose and d-desosamine. Chain extension and appropriate reduction processes lead to an enzyme-bound polyketide in which one carbonyl group has suffered total reduction, four have been reduced to alcohols, whilst one carbonyl is not reduced, and remains throughout the sequence. These processes ultimately lead to release of the modified polyketide as the macrolide ester 6-deoxyerythronolide B, a demonstrated intermediate in the pathway to erythromycins (Figure 3.31). The stereochemistry in the chain is controlled by the condensation and reduction steps during chain extension, but a reassuring feature is that there appears to be a considerable degree of stereochemical uniformity throughout the known macrolide antibiotics. In the later stages of the biosynthesis of erythromycin, hydroxylations at carbon atoms 6 and 12, and addition of sugar units, are achieved. Methylation of a sugar hydroxyl is the last step in the sequence. O

starter unit

extender units CO2H

SCoA

OH

SCoA

+ 6×

O

O

O

OH

O 1: identify starter and extender units from hypothetical poly-b-keto chain that retains all carbonyls; start by breaking lactone function and using any oxygen functions as a guide

6-deoxyerythronolide B not reduced

O

O HO

O

OH

erythronolide A

oxygens in bold are part of poly-b-keto chain OH

hydroxyls are not part of the poly-b-keto chain; they are introduced later

Figure 3.32

O O

reduced dehydrated reduced

reduced

OO OH

O

2: deduce fate of carbonyls reduced

OH OH O

OH

OH OH SEnz

O

SEnz O

O

OH reduced

OH reduced

starter unit

hypothetical poly-β-ketoester

enzyme-bound polyketide

70 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition These latter modifications illustrate that features of the biosynthesis can be considered according to the timing of the steps in the sequence. The PKS controls a number of structural modifications on the basic polyketide, especially the reductive processes, whereas others are post-PKS changes. Here, hydroxylations, glycosylations, and methylation are post-PKS changes. It is usually possible to demonstrate these later conversions by appropriate interconversions and the isolation of enzymes catalysing the individual steps. Since many macrolide structures are quite complex, and a variety of starter and extender units may be employed, it is appropriate here to demonstrate how eryA-I

eryA-II

eryA-III

DEBS 1

DEBS 2

DEBS 3

Module 1

load

simple analysis of the structure can help rationalize the biosynthetic origins of the basic macrolide. It also provides pointers to which modifications are likely to occur later in the pathway. Thus erythronolide A, the aglycone of erythromycin A, should be related to the hypothetical non-cyclized poly-β-ketoester, as shown in Figure 3.32. The positions of oxygen functions in erythronolide A are markers for the poly-β-keto system; the hydroxyls at C-6 and C-12 are not accommodated by alternate oxygenation, and can be deduced to be introduced later. Since a combination of acetyl starter and malonyl extender would give a straight-chain poly-β-ketoester, it is possible to identify the actual starter and extender

Module 3

Module 2

Module 5

Module 4

genes proteins release

Module 6

modules

ER KR AT

ACP

KS

AT

S

KR ACP

KS

AT

ACP

AT

ACP KS

KR

AT

ACP

S

S

S

O

KS

DH

KR KS

AT

KR ACP

KS

AT

S

S

S

O

O

O

HO

HO

O

HO

HO

O

HO

HO

O

HO

HO

O

HO

HO

SCoA

O

O

O

HO

HO HO

O CO2H

SCoA O SCoA

CO2H O

CO2H

HO

SCoA O

ACP: acyl carrier protein AT: acyltransferase DH: dehydratase ER: enoyl reductase KR: β-ketoacyl reductase KS: β-ketoacyl synthase TE: thioesterase bonding through phosphopantetheine

CO2H

O

SCoA O CO2H

OH O O

OH OH

6-deoxyerythronolide B

Figure 3.33

SCoA O CO2H

ACP

TE

enzyme activities

The Acetate Pathway: Fatty Acids and Polyketides units used from the extra substituents at the start and between the carbonyls. In this case, a combination of propionyl-CoA as starter and six methylmalonyl-CoA extenders can be deduced. Comparison with erythronolide A then allows the fate of the carbonyl groups during polyketide assembly to be appraised. Cyclization of this polyketide upon release from the enzyme should generate 6-deoxyerythronolide B. 6-Deoxyerythronolide B synthase (DEBS) is a modular type I PKS involved in erythromycin biosynthesis; its organization and function are illustrated in Figure 3.33. The enzyme contains three subunits (DEBS-1, DEBS-2, and DEBS-3), each encoded by a gene (eryA-I, eryA-II, and eryA-III). It has a linear sequence of six modules, each of which contains the activities needed for one cycle of chain extension, and a loading domain associated with the first module. A minimal module contains a β-ketoacyl synthase (KS), an acyltransferase (AT), and an ACP that

module n

module n-1

S

KS

AT

KR KS

ACP

SH OH

together would catalyse a two-carbon chain extension. The specificity of the AT for either malonyl-CoA or an alkyl-malonyl-CoA determines which two-carbon chain extender is used; in DEBS, the extender units are all methylmalonyl-CoA. The starter unit used is similarly determined by the specificity of the AT in the loading domain; for DEBS, this is propionyl-CoA. After each condensation reaction, the oxidation state of the β-carbon is determined by the presence of a β-ketoacyl reductase (KR), a KR plus a dehydratase (DH), or a KR plus DH plus an enoyl reductase (ER) in the appropriate module. Note that these modifications occur on the β-carbonyl, which is actually provided by the preceding module, i.e. module 1 modifies the oxidation state of the starter unit, etc. Thus, in DEBS, module 3 lacks any β-carbon-modifying domains, modules 1, 2, 5, and 6 contain KR domains and are responsible for production of hydroxy substituents, whereas module 4

CoAS

KR ACP

SH

AT

methylmalonyl transferred to next ACP

OH O

O

KR

ACP

OH

S

O

KS

AT

SH

S O R AT acylated with (2S)methylmalonyl extender O

KR ACP

R

R

transfer of growing chain from ACP to KS of next module

KS

SH

O

O

O

O

71

S O R O

OH

AT

ACP

SH

O

OH

processes repeated NADPH

KR KS

release of product in final module

AT

ACP

S

SH OH O

preceding unit modified according to other domains in module; reduction by KR shown

R final module with TE KR AT

SH OH O

KR ACP

S O

O R

KR ACP

S

TE

KS

OH

SH OH transfer to TE domain

HO R

Figure 3.34

AT

SH OH O

HO

KS

KR KS

AT

ACP

TE

HS O

O

lactonization and release from TE lactone

HO R(OH)

KS

AT

S

OH O

R O Claisen condensation reaction; growing chain attached to ACP with controlled stereochemistry

ACP

S

O H

72 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition are now catalysed according to the presence of KR, DH, and ER domains in this module. In the example shown, reduction of the ketone to a hydroxyl is catalysed by the KR domain. The ACP is linked to the chain through a phosphopantetheine group, allowing the various domains to be accessed, as seen in FASs. Transfer of the modified chain to KS in the next module allows the sequence to be repeated. Alternatively, at the end of the sequence, transfer to the TE allows lactonization (via one of the alcohol functions) and release from the enzyme. Overall, the AT specificity and the catalytic domains on each module determine the structure and stereochemistry of each two-carbon extension unit, the order of the modules specifies the sequence of the units, and the number of modules determines the size of the polyketide chain. The vast structural diversity of natural polyketides arises from combinatorial possibilities of arranging modules containing the various catalytic domains, the sequence and number of modules, the stereochemistry of associated side-chains, and the post-PKS enzymes which

contains the complete KR, DH, and ER set, and results in complete reduction to a methylene. The chain is finally terminated by a thioesterase (TE) activity which releases the polyketide from the enzyme and allows cyclization. The reactions involved in a typical module are outlined in Figure 3.34. The KS is responsible for the Claisen reaction and the growing chain is transferred to it from the ACP domain of the previous module. Initially, a starter unit, e.g. propionyl-CoA, would be bonded to the AT of the loading domain, then transferred to the loading domain’s ACP – these steps are not shown in Figure 3.34, though analogous ones form part of the sequence given. The extender unit, here methylmalonyl-CoA, is attached to the AT domain, then transferred to the next downstream ACP. At this stage, the Claisen reaction is catalysed and the growing chain is thus attached to the ACP. The resultant stereochemistry of the methyl groups is also controlled by the enzyme; only (2S)-methylmalonyl-CoA is utilized, but epimerization occurs in some cycles, changing the stereochemistry. The reduction processes, if any,

DEBS 1

DEBS 2

Module 1

load

Module 3

Module 2

DEBS 3 Module 5

Module 4

Module 6

release

ER KR AT

ACP

KS

KR

AT

ACP

KS

AT

ACP

KS

AT

ACP

KS

DH

KR

AT

ACP

KR

KR KS

AT

ACP

KS

AT

O

module 2

each module programmes the nature of the extender unit added (via AT), and the oxidation state of the b-carbon in the preceding unit (via KR, DH, ER)

module 1

Module 1

load

Module 2

KR AT

ACP

KS

AT

KS

AT

Module 1

ACP

KS

AT

Module 3

KS

AT

module 4 OH OH module 5

starter + 2 extender units

O OH

ACP

KS

truncated DEBS plus TE domain

Figure 3.35

O

release

KR ACP

module 3

TE

O

Module 2

KR AT

TE

module 6 ACP

DEBS 1 plus TE domain

load

O O

OH

KR ACP

OH

loading

release

ACP

AT

ACP

TE

H

O

O

starter + 3 extender units

The Acetate Pathway: Fatty Acids and Polyketides

Module 1

load

O Module 2

KR AT ACP

KS

AT

KR ACP

73

KS

AT

OH

ACP

H

loading domain from avermectin PKS specifies isobutyryl-CoA

O

O

OH OH

alternative starter unit O

Module 5

other modules as native DEBS

Module 6

KR KS

AT

release

KR ACP

KS

OH ACP

AT

TE

acyltransferase domain from rapamycin PKS specifies malonyl-CoA Module 5

Module 6

KR KS

AT

H

O

O

OH

one alternative extender unit O

release

KR ACP

KS

AT

ACP

OH

TE

inactivation of ketoreductase domain prevents b-carbon processing

OH

H O

O

OH O

no reduction of carbonyl

Figure 3.35 (continued) subsequently modify the first-formed product by oxidation, glycosylation, etc., e.g. 6-deoxyerythronolide B → erythromycin A. Genetic engineering now offers vast opportunities for rational modification of the resultant polyketide structure, especially in PKS enzymes of the modular type I, which prove particularly adaptable. A few representative examples of more than a hundred successful experiments leading to engineered polyketides using the DEBS protein are shown in Figure 3.35. Reducing the size of the gene sequence so that it encodes fewer modules results in the formation of smaller polyketides, characterized by the corresponding loss of extender units; in these examples, the gene encoding the chain-terminating TE also has to be attached to complete the biosynthetic sequence. Such products are naturally considerably smaller than erythromycin, but they have value as synthons containing defined functions and stereochemistry and can be used in the total synthesis of other natural product derivatives (see page 91). Replacing the loading domain of DEBS with that from another PKS, e.g. that producing avermectin (see page 80), alters the

specificity of the enzyme for the starter unit. The loading module of the avermectin-producing PKS actually has a much broader specificity than that for DEBS; Figure 3.35 shows the utilization of isobutyryl-CoA, which features in the natural biosynthesis of avermectin B1b . Other examples include the replacement of an AT domain (in DEBS specifying a methylmalonyl extender) with a malonyl-specific AT domain from the rapamycin-producing PKS (see page 84), and inactivation of a KR domain by modifying a catalytic amino acid, thus stopping any β-carbon processing for that module with consequent retention of a carbonyl group. Not all experiments in gene modification are successful, and even when they are, yields can be disappointingly lower than in the natural system. There is always a fundamental requirement that enzymes catalysing steps after the point of modification need to have sufficiently broad substrate specificities to accept and process the abnormal compounds being synthesized; this becomes more unlikely where two or more genetic changes have been made. Nevertheless,

74 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Module 1

load

KR AT

ACP

AT

KS

KS

AT

O

R

S

AT

ACP

ACP

KS

AT

KR ACP

KS

AT

ACP

S

S

S

HO

S

O

O

HO

HO

O

O

unnatural substrate analogue binds to KS of module 2 and is chain extended with methylmalonyl

Module 2

KR

KR ACP

inactivation of ketosynthase domain in module 1 prevents all normal processing

OH

Module 1

load

Module 2

HO

HO O

H N

DEBS1: normal processing

R O OH

N-acetylcysteamine thioester

HS

R

H N

O

H

OH

O

Coenzyme A

OH

O

Figure 3.36

CO2H starter

O

E1

CO2H SCoA

st

OH SEnz O

8a

epoxidation

3 OH

+

O

8

1

O

6×

O

2

SCoA

6

4 5

O2 NADPH

OH E1

OH

O O

OH

oleandolide

E2

O

TDP-L-olivose

E3

O L-olivosyl-oleandolide

8,8a-deoxyoleandolide

modules 1−6

O

SAM O-methylation of olivose

E4

O L-oleandrosyl-oleandolide E5

OH O E1: oleandomycin PKS O (OlePKS; OleA1−3) E2: OleP oleandolide E3: OleG2 (olivosyl transferase) E4: OleY (methyltransferase) E5: OleG1 (desosaminyl transferase)

O

HO

O

TDP-D-desosamine

O D-desosamine

O

OMe OH L-oleandrose

oleandomycin

Figure 3.37

NMe2

OH O

OH OH L-olivose

The Acetate Pathway: Fatty Acids and Polyketides some multiple modifications have been successful, and it has also been possible to exploit changes in a combinatorial fashion using different expression vectors for the individual subunits, thus creating a library of polyketides, which may then be screened for potential biological activity. Another approach is to disable a particular enzymic function, again by altering a specific amino acid, and to supply an alternative synthetic substrate analogue. Provided this substrate is not too dissimilar to the natural substrate, it may then be processed by the later enzymes. This is exemplified in Figure 3.36, where a number of artifical substrates may be utilized to produce a range of erythromycin derivatives inaccessible by semi-synthesis.

The KS domain in module 1 is disabled by replacement of the active site cysteine, and the alternative substrate is supplied in the form of an N-acetylcysteamine thioester. The N-acetylcysteamine group acts as a simple analogue of coenzyme A, which is acceptable to and processed by the PKS. A combination of propionate and acetate units is used to produce the 14-membered macrocyclic ring of oleandomycin (Figure 3.37) from Streptomyces antibioticus [Box 3.6], but otherwise, many of the structural features and the stereochemistry of oleandomycin resemble those of erythromycin A. Structural analysis as described above for erthyromycin would predict that the starter unit should

module n KS

S

AT

load KSQ

ACP

standard decarboxylation and Claisen condensation reaction catalysed by KS

OH S O

O

75

AT

load KSQ

ACP

OH S O

R

ACP

OH S O only decarboxylation step catalysed by KSQ

H O

O

AT

O H

O H

Figure 3.38 starter + modules 1−3, 7

module 4

CO2H SCoA

5×

module 5

CO2H SCoA

+

O

module 6 CO2H

CO2H

O

SCoA

+

+ MeO

SCoA

O methoxymalonyl-CoA

O ethylmalonyl-CoA

E1 O

Me2N OH

O

O

3

st

O

4

2 1

D-forosamine

NMe2

5 MeO O

OH EnzS 7

6

OH

MeO O

E1

OH

O

E1: platenolide synthase (Srm1−5)

Figure 3.39

OH OH

platenolide

MeO O O

HO O OR

O

O

D-mycaminose

R = H, leuconolide A1 R = H, spiramycin I R = COCH3, spiramycin II R = COCH2CH3, spiramycin III

OH

O

OH L-mycarose

76 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition be acetyl-CoA, whilst six propionates, via methylmalonyl-CoA, supply the extension units (Figure 3.37). The oleandomycin PKS (OlePKS) is organized essentially the same as DEBS, though with one significant difference. It has been discovered that the loading domain in the OlePKS contains an AT specific for malonyl-CoA (not acetyl-CoA) and contains an additional KS domain (KSQ ). In KSQ , an active site Cys residue is replaced by Gln and cannot make the essential thioester linkage; Q is the single-letter abbreviation for glutamine (see page 423). Consequently, the KSQ domain cannot participate in any Claisen reaction, and its role is to decarboxylate the ACP-bound substrate to acetyl-ACP for use as the starter (Figure 3.38); many other examples with this type of mechanism are known. Hence, oleandomycin is derived from malonyl-CoA and six methylmalonyl-CoAs. The 8-methyl group is subsequently modified by a P-450 monooxygenase to give an epoxide function. The sugar units in oleandomycin are d-desosamine (as in erythromycin) and l-oleandrose; as with erythromycin, O-methylation of one sugar unit occurs after attachment to the macrolide. Spiramycin I (Figure 3.39) [Box 3.6] from Streptomyces ambofaciens has a 16-membered lactone ring and is built up from a combination of six acetate units (one as starter, but all derived from malonyl-CoA), one propionate extender (from methylmalonyl-CoA), together starter + modules 1, 2, 4, 6

modules 3, 7

SCoA

5×

module 5

CO2H

CO2H

3 SCoA

+

4

2

O

O

O

CO2H SCoA

+ 2×

with two further variants. First, a butyrate chain extender is incorporated via ethylmalonyl-CoA and yields an extension unit having an ethyl side-chain. Second, a methoxymalonyl-CoA extender incorporates an extension unit with a methoxy side-chain; this function might plausibly be expected to arise from hydroxylation followed by methylation. The relationships are outlined in Figure 3.39; the first-formed product is platenolide. The platenolide PKS is composed of seven modules plus a loading domain, organized into five subunits: loading domain plus modules 1 and 2 on the first subunit, module 3 on the second, modules 4 and 5 on subunit 3, and modules 6 and 7 on subunits 4 and 5. Platenolide can be modified to a number of other macrolides. For the spiramycins, the ethyl side-chain is subsequently oxidized to generate an aldehyde. Spiramycin I also contains a conjugated diene, the result of carbonyl reductions being followed by dehydration during chain assembly. Tylosin (Figure 3.40) [Box 3.6] from Streptomyces fradiae has many structural resemblances to the spiramycins, but can be analysed as a propionate starter (actually derived from methylmalonyl-CoA) with chain extension from two malonyl-CoA units, four methylmalonyl-CoA units, and one ethylmalonyl-CoA unit. Oxidation at C-20 in the ethyl side-chain to give an aldehyde is known to occur after introduction of the first sugar, d-mycaminose, and this is followed by hydroxylation of the methyl C-23; both enzymes involved are cytochrome P-450 systems.

O

5

1

E1

O

st

OH EnzS 7

OH

6

OH

O

O E1

O NMe2

O

HO

O

HO O

O

OMe

OMe D-mycinose

O tylonolide

OH

tylosin E1: tylactone PKS (Tyl1−5)

Figure 3.40

20

O

D-mycaminose

O

23

OH

O O L-mycarose

OH

O tylactone

OH OH

The Acetate Pathway: Fatty Acids and Polyketides

77

Box 3.6 Macrolide Antibiotics The macrolide antibiotics are macrocyclic lactones with a ring size typically 12–16 atoms, and with extensive branching through methyl substituents. Two or more sugar units are attached through glycoside linkages; these sugars tend to be unusual 6-deoxy structures often restricted to this class of compounds. Examples include l-cladinose, l-mycarose, d-mycinose, and l-oleandrose. At least one sugar is an amino sugar, e.g. d-desosamine, d-forosamine, and d-mycaminose. These antibiotics have a narrow spectrum of antibacterial activity, principally against Gram-positive microorganisms. Their antibacterial spectrum resembles, but is not identical to, that of the penicillins, so they provide a valuable alternative for patients allergic to the penicillins. Erythromycin is the principal macrolide antibacterial currently used in medicine.

Erythromycins The erythromycins (Figure 3.41) are macrolide antibiotics produced by cultures of Saccharopolyspora erythraea (formerly Streptomyces erythreus). The commercial product erythromycin is a mixture containing principally erythromycin A plus small amounts of erythromycins B and C (Figure 3.41). Erythromycin activity is predominantly against Gram-positive bacteria, and the antibiotic is prescribed for penicillin-allergic patients. It is also used against penicillin-resistant Staphylococcus strains, in the treatment of respiratory tract infections, and systemically for skin conditions such as acne. It is the antibiotic of choice for infections of Legionella pneumophila, the cause of legionnaire’s disease. Erythromycin exerts its antibacterial action by inhibiting protein biosynthesis in sensitive organisms. It binds reversibly to the larger 50S subunit of bacterial ribosomes and blocks the translocation step in which the growing peptidyl-tRNA moves from the aminoacyl acceptor site to the peptidyl donor site on the ribosome (see page 422). The antibiotic is a relatively safe drug with few serious side-effects. Nausea and vomiting may occur, and if high doses are prescribed, a temporary loss of hearing might be experienced. Hepatotoxicity may also occur at high dosage. Unfortunately, the drug has a particularly vile taste. Erythromycin is unstable under acidic conditions, undergoing degradation to inactive compounds by a process initiated by the 6-hydroxyl attacking the 9-carbonyl to form a hemiketal, and by a similar reaction involving the 12-hydroxyl. Dehydration to an enol ether may follow, though the principal product is the spiroketal anhydroerythromycin A (Figure 3.42). The keto form is the only material with significant antibacterial activity, and the enol ether shown is responsible for gastrointestinal side-effects. Thus, to protect oral preparations of erythromycin against gastric acid, they are formulated as enteric coated tablets, or as insoluble esters (e.g. ethyl succinate esters) which are then hydrolysed in the intestine. Esterification produces a taste-free product, and typically involves the hydroxyl of the amino sugar desosamine. To reduce acid instability, semi-synthetic analogues of erythromycin have also been developed. Clarithromycin (Figure 3.41) is a 6-O-methyl derivative of erythromycin A; this modification blocks 6,9-hemiketal formation as in Figure 3.42. Azithromycin (Figure 3.41) is a ring-expanded aza-macrolide in which the carbonyl function has been removed. In both analogues, the changes enhance activity compared with that of erythromycin, and cause fewer gastrointestinal side-effects. Clarithromycin is used as part of the drug regimen for eradication of Helicobacter pylori in the treatment of gastric and duodenal ulcers. O

Me

O

N R1

OH

D-desosamine

OH HO O O

HO

O O

HO

OH O

2

O OH R2 = H, L-mycarose R2 = Me, L-cladinose

R1 = OH, R2 = Me, erythromycin A R1 = H, R2 = Me, erythromycin B R1 = OH, R2 = H, erythromycin C

OH

O O

NMe2 HO

O

O

OMe

O

D-desosamine

OH

NMe2 HO

O OR

O

Figure 3.41

OMe

NMe2

O OH

O O

O OMe

O

OH L-cladinose

clarithromycin (6-O-methyl erythromycin A)

azithromycin

78 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.6 (continued) Bacterial resistance to erythromycin has become significant and has limited its therapeutic use against many strains of Staphylococcus. Several mechanisms of resistance have been implicated, one of which is a change in permeability of the bacterial cell wall. Differences in permeability also appear to explain the relative insensitivity of Gram-negative bacteria to erythromycin when compared with Gram-positive bacteria. Resistant bacteria may also modify the chemical nature of the binding site on the ribosome, thus preventing antibiotic binding, and some organisms are now known to metabolize the macrolide ring to yield inactive products. New derivatives based upon 3-keto-6-O-methylerythromycin A, termed ketolides, are providing important advances in this respect. These agents retain potent antibacterial activity towards erythromycin-susceptible organisms, but improved activity towards MLSB (macrolide–lincosamide–streptogramin B)-resistant bacteria. In general, the ketolide structures differ from erythromycin by removal of the cladinose sugar and replacement with a 3-keto group, the presence of a 6-methoxy group, and attaching a cyclic carbamate at C-11/C-12. The first of these drugs in use is telithromycin (Figure 3.43).

O 9

HO

OH

OH OH

12

HO O

O O

dehydration H

H2O

H+

NMe2

6

O

acid-catalysed formation of hemiketal OH OH

O

O

O OMe

6,9-hemiketal – H2O

erythromycin A

OH

O

OH 9

HO 12

HO

O O

NMe2 HO O

O O

O

anhydroerythromycin A

O

O

6

O

enol ether

alternative 9,12-hemiketal

OMe OH

O

Figure 3.42

O N-substituted 11,12-carbamate ring O

11

NR

12

NMe2

6

O O O

O

6-O-methyl OMe substituent

HO O 3

OMe

O

N

N

N

O

NMe2

O O

HO O

O

O 3-keto group

ketolides

N

O

O telithromycin

Figure 3.43

Oleandomycin and Spiramycin Oleandomycin (Figure 3.37) is produced by fermentation cultures of Streptomyces antibioticus and has been used medicinally as its triacetyl ester troleandomycin against Gram-positive bacterial infections; it is rarely employed now. The spiramycins

The Acetate Pathway: Fatty Acids and Polyketides

79

Box 3.6 (continued) (Figure 3.39) are macrolides produced by cultures of Streptomyces ambofaciens. The commercial antibiotic is a mixture containing principally spiramycin I, together with smaller amounts (10–15% each) of the acetyl ester spiramycin II and the propionyl ester spiramycin III. This antibiotic has recently been introduced into medicine for the treatment of toxoplasmosis, infections caused by the protozoan Toxoplasma gondii.

Tylosin Tylosin (Figure 3.40) is an important veterinary antibiotic. It is produced by Streptomyces fradiae, and is used to control chronic respiratory diseases caused by Mycoplasma galliseptum in poultry and to treat Gram-positive infections in pigs.

starter

modules 2−5, 8, 10, 12

modules 1, 6, 7, 9, 11

SCoA

SCoA

+ 7×

O

1

CO2H

CO2H

HO

SCoA

+ 5×

4 E1

O

O

OH O

5

6

2 HO

st

3

7

HO SEnz 12 O 11

O 8 CO2H NH2 L-Ile

HO

O

O NADPH E3

5

5

O

H

H

OH avermectin B1a aglycone

2 3

6

E2

5

4

O

O

O

O

TDP-L-oleandrose O

TDP-L-oleandrose E4 H

22

O

MeO

O

L-oleandrose

O

HO

MeO L-oleandrose O avermectin B1a

25

SEnz

O

B O

O

O

O

H

aldol reaction

23

O

O

enolate anion formation O O

SEnz

6,8a-seco-6,8a-deoxy5-oxoavermectin aglycone E4

HO

O

H

O spiro-ketal

HO

O2 NADPH 8a

O

10 O

HO

HO

9

E1

H

SEnz

HO

H

avermectin B1a aglycone

O

5

H

OH 23 22

E1: E2: E3: E4:

avermectin PKS (aveA; AVES1−4) aveE aveF aveB (oleandrosyl transferase)

CO2H NH2 L-Val

Figure 3.44

SCoA

25

O O

H

avermectin B1b

80 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition The avermectins (Figure 3.44) have no antibacterial activity, but possess anthelmintic, insecticidal, and acaricidal properties, and these are exploited in human and veterinary medicine [Box 3.7]. The avermectins are also 16-membered macrolides, but their structures are made up from a much longer polyketide chain (starter plus 12 extenders) which is also used to form oxygen heterocycles fused to the macrolide. Avermectin B1a exemplifies a typical structure, and the basic carbon skeleton required to produce this can be postulated as in Figure 3.44. The starter unit in this case would be 2-methylbutyryl-CoA, which is derived from the amino acid l-isoleucine (compare iso-fatty acids, page 56). Both malonyl-CoA and methylmalonyl-CoA are then utilized as extender units. The simple structural analysis approach does not pick up two variants: extender 10 contains an unmodified keto function, and there is a double bond between extenders 11 and 12, not present in the final structure (C-2,3 in avermectin). Further, the product of the avermectin PKS is not a simple macrolide, but a structure in which additional

cyclizations have also been catalysed. The spiroketal system is easily accounted for as a combination of ketone and two alcohol functions. The cyclohexenone ring arises by an aldol reaction as shown, and is dependent upon the 2,3-double bond and 5-keto function just mentioned. This reaction may precede lactone ring formation, based on the increased acidity of thioesters relative to oxygen lactones. The avermectin PKS is composed of four polypeptide subunits: load plus modules 1 and 2; modules 3–6; modules 7–9; modules 10–12. Intriguingly, module 7 contains a DH domain and module 10 a KR domain, but both of these are non-functional, so do not contribute to the oxygenation state of the extender unit added; in the other systems discussed so far, variation in extender modification is simply a result of the appropriate domain being absent. Post-PKS modifications include oxidative cyclization to produce the tetrahydrofuran ring, which appears to be catalysed by a single cytochrome P-450-dependent enzyme. Glycosylation is accomplished, unusually, by the repeated action of a single glycosyltransferase.

Box 3.7 Avermectins The avermectins are a group of macrolides with strong anthelmintic, insecticidal, and acaricidal properties, but with low toxicity to animals and humans. They are produced by cultures of Streptomyces avermectilis. Some eight closely related structures have been identified, with avermectins B1a (Figure 3.44) and B2a (Figure 3.45) being the most active antiparasitic compounds. Abamectin (a mixture of about 85% avermectin B1a and about 15% avermectin B1b ) is used on agricultural crops to control mites and insects. Ivermectin (Figure 3.45) is a semi-synthetic 22,23-dihydro derivative of avermectin B1a (the commercial product also contains up to 20% dihydroavermectin B1b ) and was first used in veterinary practice against insects, ticks, mites, and roundworms. Although it is a broad spectrum nematocide against roundworms, it is inactive against tapeworms and flatworms, or against bacteria and fungi. It is an extremely potent agent, and is effective at very low dosages. It is now the drug of choice for use against filarial and several other worm parasites in humans, e.g. river blindness caused by the nematode Onchocerca volvulus. The avermectins and ivermectin target the glutamate-gated chloride channels unique to nematodes, insects, ticks, and arachnids, resulting in neuro-muscular paralysis and death. 23

22

H

O

O O

O O

O O

H

MeO

O

O

avermectin B1a

HO

H

O

HO

MeO OH 23

22

H

avermectin B2a

doramectin O

H

ivermectin

HO

5

O

O O

Figure 3.45

23 22

O

H

OH 5

O H

selamectin

N

OH

The Acetate Pathway: Fatty Acids and Polyketides

81

Box 3.7 (continued) Doramectin is a newer analogue of avermectin B1a (Figure 3.45) with extended biological activity, used to protect cattle against internal and external parasites. It is produced in a mutant of Streptomyces avermectilis that is unable to synthesize the branched-chain starter acids required for avermectin biosynthesis. The organism is able to utilize exogenously supplied acids as starter groups and incorporate them into modified avermectins; doramectin is produced by the use of cyclohexanecarboxylic acid in the culture medium (compare penicillins, page 461). It has also proved possible to express in this mutant biosynthetic genes for the production of cyclohexanecarbonyl-CoA, a natural compound involved in the biosynthesis of some other antibiotics. This engineered mutant synthesizes doramectin without the need for cyclohexanecarboxylic acid supplementation. Selamectin is the 5-oxime of doramectin, used to control fleas and internal parasites in dogs and cats.

Avermectins are usually isolated as a mixture in which the main a component has a 2-butyl group (derived from isoleucine) at C-25, whilst the minor b component has a 2-propyl group instead, e.g. avermectin B1b . In this case, the starter group is 2-methylpropionyl-CoA, derived from the amino acid l-valine. The A-series of avermectins are the 5-methoxy analogues of the B-series. Even larger macrolides are encountered in the polyene macrolides, most of which have antifungal properties, but not antibacterial activity [Box 3.8]. The macrolide ring size ranges from 26 to 38 atoms, and this also accommodates a conjugated polyene of up to seven E double bonds. Relatively few methyl groups are attached to the ring, and thus malonyl-CoA is utilized more frequently than methylmalonyl-CoA as chain extender. Typical examples are amphotericin B (Figure 3.46) from Streptomyces nodosus and nystatin A1 from Streptomyces noursei. These

have very similar structures and are derived from the same basic precursors (Figure 3.46). The ring size becomes contracted due to cross-linking by formation of a hemiketal; this modification occurs before or during release from the PKS. The two compounds have slightly different hydroxylation patterns, part of which is introduced by a post-PKS hydroxylation. The two areas of conjugation in nystatin A1 are extended into a heptaene system in amphotericin B. Both compounds are carboxylic acids, a result of oxidation of a propionate-derived methyl group, and glycosylated with the amino sugar d-mycosamine. The PKSs involved in amphotericin and nystatin biosynthesis are also closely related. They are composed of six polypeptide subunits: load; modules 1 and 2; modules 3–8; modules 9–14; modules 15–17; module 18. Both proteins also contain some non-functional domains.

Box 3.8 Polyene Antifungals The polyene antifungals are a group of macrocyclic lactones with very large 26–38-membered rings. They are characterized by the presence of a series of conjugated E double bonds and are classified according to the longest conjugated chain present. Medicinally important ones include the heptaene amphotericin B and the tetraene nystatin. There are relatively few methyl branches in the macrocyclic chain. The polyenes have no antibacterial activity but are useful antifungal agents. Their activity is a result of binding to sterols in the eukaryotic cell membrane; this action explains the lack of antibacterial activity, because bacterial cells do not contain sterol components (see page 241). Fungal cells are also attacked rather than mammalian cells, since the antibiotics bind about 10-fold more strongly to ergosterol, the major fungal sterol (see page 252), than to cholesterol, the main animal sterol component (see page 251). This binding modifies the cell wall permeability and leads to formation of transmembrane pores that allow K+ ions, sugars, and proteins to be lost from the microorganism. Though binding to cholesterol is less than to ergosterol, it is responsible for the observed toxic side-effects of these agents on humans. The polyenes are relatively unstable, undergoing light-catalysed decomposition, and are effectively insoluble in water. This insolubility actually protects the antibiotic from gastric decomposition, allowing oral treatment of infections in the intestinal tract. Amphotericin is an antifungal polyene produced by cultures of Streptomyces nodosus and contains principally the heptaene amphotericin B (Figure 3.46) together with structurally related compounds, e.g. the tetraene amphotericin A (about 10%) which is the 28,29-dihydro analogue of amphotericin B. Amphotericin A is much less active than amphotericin B. Amphotericin is active against most fungi and yeasts, but it is not absorbed from the gut, so oral administration is restricted to the treatment of intestinal candidiasis. It is administered intravenously for treating potentially life-threatening systemic fungal infections. However, it then becomes highly protein bound, resulting in poor penetration and slow elimination from the body. After parenteral administration, toxic side-effects, including nephrotoxicity, are relatively common. Close supervision and monitoring of the patient is thus

82 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.8 (continued) necessary, especially since the treatment may need to be prolonged. Lipid formulations of amphotericin are much less toxic and have proven a significant advance. Candida infections in the mouth or on the skin may be treated with appropriate formulations. More recently, amphotericin has been shown to be among the few agents that can slow the course of prion disease in animal models. Nystatin is a mixture of tetraene antifungals produced by cultures of Streptomyces noursei. The principal component is nystatin A1 (Figure 3.46), but the commercial material also contains nystatin A2 and A3 ; these have additional glycoside residues. Nystatin is too toxic for intravenous use, but has value for oral treatment of intestinal candidiasis, as lozenges for oral infections, and as creams for topical control of Candida species.

CO2H

CO2H SCoA + 15 × O

SCoA

SCoA + 3 × O

O E1

EnzS OH O

HO

E5

OH OH

OH

OH

OH

O HO EnzS OH

OH E1

hemiketal

OH

8

O

OH O

HO

OH

OH

OH

OH

OH

OH E5

HO

O

OH

OH

OH

OH

O

O2 NADPH

OH

O

OH

OH

OH

OH

OH

O

28

OH

OH

OH

OH

O

CO2H

hydroxylation at C-10; oxidation of 16-methyl; E6, E7 order not known E8

O2 NADPH GDP-D-mycosamine

HO HO H2N

O O

O HO

O

OH

OH

OH

OH

OH O

D-mycosamine amphotericin B nystatinolide

E1: E2: E3: E4:

amphotericin PKS (AmphA−C, AmphI−K) AmphL AmphN AmphDI (mycosaminyl transferase)

Figure 3.46

OH 16

OH OH

OH

29

amphoteronolide A

O

HO

OH

10

GDP-D-mycosamine

O HO

hemiketal

16

O hydroxylation at C-8; oxidation of 16-methyl; E2, E3 order not known E4

O HO

E5: E6: E7: E8:

nystatin PKS (NysA−C, NysI−K) NysL NysN NysDI (mycosaminyl transferase)

HO HO H2N

O

D-mycosamine nystatin A1

O

OH CO2H

The Acetate Pathway: Fatty Acids and Polyketides HO

4

HO

5

OH

SCoA

+ 2×

O dihydroxycyclohexenecarboxlic acid

OH

SCoA

+ 5× O

O

SCoA

+

+

O ethylmalonyl-CoA CO2H

SCoA

+ 2 × MeO

O methoxymalonyl-CoA

CO2H

CO2H

HO HO

CO2H

CO2H

CO2H

83

NH pipecolic acid (from lysine)

E1

shikimic acid

HO

HO cyclohexene ring is reduced during HO polyketide formation

O OH

HO

OH

O 31

H

O

OH

EnzS

O

O

H

O

N E1: ascomycin (FK520) PKS (FkbB, FkbC, FkbA, FkbP) E2: FkbD (C-9 hydroxylase) E3: FkbO (C-9 hydroxyl oxidase) E4: FkbM (O-methyltransferase)

N

E1 O

O OH

OH 9

OMe

O H

hemiketal

OMe

OMe

OMe

9-deoxo-31-O-demethylascomycin hydroxylation and oxidation at C-9; methylation of C-31 hydroxyl CO2H

E2, E3, E4

HO

HO SCoA

O propylmalonyl-CoA

22

MeO

H

O

O

MeO

21

OH

20

O H

O

O

O H

H O 21 20

hydroxylation dehydration

O

N

21

OH

17

N

OMe

O

O OH

O

OH

O H

O H

OMe

OMe

OMe tacrolimus (FK506)

O

ascomycin (FK520)

Figure 3.47 Two unusual and clinically significant macrolides are ascomycin (FK520), from Streptomyces hygroscopicus var. ascomyceticus, and tacrolimus (FK506), isolated from Streptomyces tsukubaensis (Figure 3.47). These compounds contain a 23-membered macrolactone that also incorporates an N-heterocyclic ring and are identical apart from the substituent at C-21. The piperidine ring and adjacent carbonyl are incorporated as pipecolic acid (see page 330) via an amide linkage onto the end of

the growing chain. Chain assembly is catalysed by an enzyme combination that is partly a PKS and partly a non-ribosomal peptide synthetase (NRPS). Though PKSs and NRPSs utilize different substrates (carboxylic thioesters and amino acid thioesters respectively), they exhibit remarkable similarities in the modular arrangement of catalytic domains and product assembly mechanism (see page 438). PKS–NRPS hybrid systems appear to have evolved, and these produce metabolites

84 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition incorporating amino acid units as well as carboxylic acids. In the cases of ascomycin and tacrolimus, the predominantly PKS system also contains an NRPS-like protein, responsible for incorporating the pipecolic acid moiety. The main chains are derived principally from acetate and propionate, the fragments of which can readily be identified. Two methoxymalonyl-CoA extenders are utilized for ascomycin production and most likely for tacrolimus as well. Methoxymalonyl-CoA is also an extender in spiramycin biosynthesis (see page 75), and its production from a C3 glycolytic pathway intermediate such as glyceric acid is also part of the PKS system. Ethylmalonyl-CoA (for ascomycin) or propylmalonyl-CoA (for tacrolimus) provides the remaining extender. In the case of tacrolimus, post-PKS hydroxylation and dehydration accounts for the allyl side-chain at C-21. The starter unit is 4,5-dihydroxycyclohex-1-enecarboxylic acid, a reduction product from shikimate; it

CO2H

HO +

OH

HO

is incorporated as the free acid, perhaps by an AMP-activated form analogous to that used in the NRPS mechanism (see page 440). A further surprise is that the cyclohexene ring is reduced during transfer from the load domain to module 1. Methylation of the 5-hydroxyl is a post-PKS modification. Although rapamycin (sirolimus) (Figure 3.48) contains a very large 31-membered macrocycle, several portions of the structure are identical to those in ascomycin and tacrolimus. Dihydroxycyclohexenecarboxylic acid and pipecolic acid are again utilized in its formation, whilst the rest of the skeleton is supplied by simple acetate and propionate residues. Methoxymalonyl-CoA is not used as an extender, and post-PKS modifications include the more common hydroxylation/methylation sequences. Ascomycin, tacrolimus, and derivatives are effective immunosuppressants, and they are proving to be valuable drugs in organ transplant surgery and in the treatment of skin problems [Box 3.9].

HO

NH

O OH

HO

O

OH EnzS

pipecolic acid

dihydroxycyclohexenecarboxlic acid

O

E1 N

CO2H

CO2H SCoA

+ 7×

O

HO

O

SCoA

+ 7× O

O

O OH

E1

OMe HO HO

O

HO OH

H

O

MeO O

OH H

O

O2 NADPH SAM

O H N

O

O H

O OH

E2−6

HO O H

further hydroxylations and methylations at indicated positions

N

O MeO OH

O

O H OMe

pre-rapamycin E1: rapamycin PKS (rapA−C, rapP) E2, E3: rapJ, rapN (hydroxylases) E4−6: rapI, rapM, rapQ (methyltransferases)

Figure 3.48

rapamycin (sirolimus)

O

The Acetate Pathway: Fatty Acids and Polyketides

85

Box 3.9 Tacrolimus and Sirolimus Tacrolimus (FK-506) (Figure 3.47) is a macrolide immunosuppressant isolated from cultures of Streptomyces tsukubaensis. It is used in liver and kidney transplant surgery. Despite the significant structural differences between tacrolimus and the cyclic peptide cyclosporin A (ciclosporin; see page 454), these two agents have a similar mode of action. They both inhibit T-cell activation in the immunosuppressive mechanism by binding first to a receptor protein giving a complex which then inhibits a phosphatase enzyme called calcineurin. The resultant aberrant phosphorylation reactions prevent appropriate gene transcription and subsequent T-cell activation. Structural similarities between the region C-17 to C-22 in tacrolimus and fragments of the cyclosporin A peptide chain have been postulated to account for this binding. Tacrolimus is up to 100 times more potent than cyclosporin A; it produces similar side-effects, including neurotoxicity and nephrotoxicity. Tacrolimus is also used topically to treat moderate to severe atopic eczema; the ascomycin derivative pimecrolimus (Figure 3.49) is also used for mild to moderate conditions. Sirolimus (rapamycin) (Figure 3.48) is produced by cultures of Streptomyces hygroscopicus and is also used as an immunosuppressant drug. Although tacrolimus and sirolimus possess a common structural unit, and both inhibit T-cell activation, they appear to achieve this by somewhat different mechanisms. The first-formed sirolimus–receptor protein binds not to calcineurin, but to a different protein. It exerts its action later in the cell cycle by blocking growth-factor-driven cell proliferation. Everolimus (Figure 3.49) is a semi-synthetic derivative of sirolimus with better oral bioavailability now available for kidney and heart transplants. Zotarolimus (Figure 3.49) inhibits cell proliferation, preventing scar tissue formation following cardiovascular surgery; this agent is delivered via a coronary stent. Some semi-synthetic sirolimus derivatives have also been investigated as anticancer drugs. These also display modifications to the C-40 hydroxyl group in the side-chain cyclohexane ring. Temsirolimus is now approved for treatment of renal cell carcinoma, and deforolimus is in advanced clinical trials against a variety of cancers (Figure 3.49).

rapamycin derivatives HO

40

MeO

39

MeO

MeO

H OH

OH

H everolimus

O N

HO

O

HO

rapamycin (sirolimus)

N N

ascomycin derivatives

P

O

H zotarolimus

MeO

O

O

H temsirolimus

MeO

H ascomycin

Cl

N MeO

32

H deforolimus

MeO

H pimecrolimus

Figure 3.49

Attracting considerable interest at the present time are the epothilones (Figure 3.50), a group of macrolides produced by cultures of the myxobacterium Sorangium cellulosum. They are formed by a multienzyme system composed of nine PKS modules and one NRPS module distributed over six protein subunits. These compounds appear to employ an unusual starter

unit containing a thiazole ring; this is constructed from the amino acid cysteine and an acetate unit by EpoB, the NRPS component of the enzyme (see also similar ring systems in bacitracin, page 443, and bleomycin, page 449). The loading module EpoA contains a decarboxylating KS domain, and the acetyl starter is thus derived from malonyl-CoA

86 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition R

S

CO2H +

SCoA

N O

CO2H +

SCoA

S

SCoA

O

E1

O

O

12

S O2 OH NADPH

N

OH

O

methylation during PKS assembly

OH

N O

4

OH

O

E1

E2

O O

OH SEnz

R

R

S

OH

N

O

O

OH

O O

R = H, epothilone C R = Me, epothilone D

R = H, epothilone A R = Me, epothilone B

O

RCH2

SEnz Ad

E1: epothilone synthetase (Epo NRPS-PKS; EpoA−F) E2: EpoK (epoxidase)

H3C

S SAM

EpoA load KSY

AT

ER

EpoB NRPS ACP

A

Cy

Ox

C-alkylation with SAM

PCP

S

Cy

A

Ox

S O

PCP

Cy

A

S O

H2N

O

N dehydration

CO2H

S

HO oxidation

KSY: KS (decarboxylating) NRPS domains: A: adenylation Cy: cyclization Ox: oxidase

L-Cys

O

S

O HS

HS

SCoA

PCP

S

H N

HN HS

Ox

O cyclization

CO2H

A

S

O H2N

Cy

PCP

Ox

Cy

A

Ox

PCP

S O

O

N

O

S

S OH

N

S OH

N

HN

O O

OH

O

ixabepilone

O

OH

O

sagopilone

Figure 3.50 (compare oleandomycin, page 73). The macrolide ring also contains an extra methyl group at C-4, the result of methylation during polyketide chain assembly by a methyltransferase domain in module 8, part of the EpoE protein; this undoubtedly occurs whilst a β-diketone

function is present. The other interesting feature is that this bacterium produces epothilone A and epothilone B in a ratio of about 2:1. These compounds differ in the nature of the substituent at C-12, which is hydrogen in epothilone A but a methyl group in epothilone B.

The Acetate Pathway: Fatty Acids and Polyketides Genetic evidence shows that the PKS enzyme can accept either malonyl-CoA or methylmalonyl-CoA extender units for this position. Thus, epothilone B is constructed from three malonate and five methylmalonate extender units, whilst epothiolone A requires four units of each type. The first-formed products are epothilones C and D; these alkenes are converted into epoxides by a post-PKS cytochrome P-450-dependent epoxidase activity. The

CO2H

epothilones display marked antitumour properties with a mode of action paralleling that of the highly successful anticancer drug taxol (see page 224). However, the epothilones have a much higher potency (2000–5000 times) and are active against cell lines which are resistant to taxol and other drugs. There appears to be considerable potential for developing the epothilones or analogues into valuable anticancer drugs, and several derivatives

CO2H

PO O

CO2H

HO

OH DAHP

NH2

E3 O

OH aminoDAHP

NH2

O HO

OH aminodehydroshikimic acid

SCoA

+ 2×

– H 2O E1 E2

OH

CO2H

CO2H

PO O

HO

NH2

CO2H SCoA

+ 8×

3-amino-5-hydroxybenzoic acid

O

chain extension 3× OH

E4

aromatic hydroxylation

OH

O O

HO

NH2

EnzS NH2

O

O O

OH

EnzS

NH2

nucleophilic attack of enolate onto enone; enolization; EnzS oxidation of quinol to quinone

O O

OH

O

NH2 O O

O

O

oxidation of quinol to quinone OH

OH

O

OH

EnzS

O

OH

HO

O O

OH

EnzS

postulated polyketide

OH

NH2

chain extension; lactamization

HO O

O O

HO

HO

E4, E5

OH OH O H N

OH 34a

7

8

O

HO O

O

HO

O MeO

OH OH

several steps

OH OH

H N O

O O

O O

rifamycin B

Figure 3.51

CO2H

HO carbon lost

O

proansamycin X

OH OH OH O H N

HO

87

oxidation to 7,8-ene; hydroxylation C-34a

O

HO O

O

rifamycin W

E1: E2: E3: E4: E5:

aminodehydroquinate synthase (RifG) aminodehydroquinate dehydratase (RifJ) AHBA synthase (RifK) rifamycin synthetase (RifA−E) RifF (amide synthetase)

88 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition are undergoing clinical trials. These include epothilone B (patupilone) and its allyl analogue sagopilone; the lactam ixabepilone has recently been approved for drug use in the treatment of breast cancer (Figure 3.50). A further group of macrolides are termed ansa macrolides; these have non-adjacent positions on an aromatic ring bridged by a long aliphatic chain (Latin: ansa = handle). The aromatic portion may be a substituted naphthalene or naphthaquinone, or alternatively a substituted benzene ring. The macrocycle in the ansamycins is closed by an amide rather than an ester linkage, i.e. ansamycins are lactams. The only ansamycins currently used therapeutically are rifamycins [Box 3.10], semi-synthetic naphthalene-based macrocycles produced from rifamycin B (Figure 3.51). Rifamycin synthetase comprises a sequence of five proteins, with an NRPS-like loading module and 10 PKS elongation modules. The nitrogen of the lactam ring derives from the starter unit, 3-amino-5-hydroxybenzoic acid (Figure 3.51), which is a simple phenolic acid

derivative produced by an unusual variant of the shikimate pathway (see Chapter 4). AminoDAHP is formed via aminosugar metabolism, and then the standard shikimate pathway continues with amino analogues. 3-Amino-5-hydroxybenzoic acid is produced from aminodehydroshikimic acid by dehydration. In the biosynthesis of rifamycin B (Figure 3.51) in Amycolatopsis mediterranei, this starter unit plus two malonyl-CoA and eight methylmalonyl-CoA extenders are employed to fabricate proansamycin X as the first product released from the enzyme. Simple structural analysis might predict an enzyme-bound intermediate as shown in Figure 3.51; it is now known that the naphthoquinone ring system is constructed during chain assembly, and a proposed mechansim is outlined. Rifamycin W and then the antibiotic rifamycin B are the result of several further modifications, though much of the detail requires further investigation. Post-PKS modifications include cleavage of the double bond, loss of one carbon, and formation of the ketal.

Box 3.10 Rifamycins The rifamycins are ansamycin antibiotics produced by cultures of Amycolatopsis mediterranei (formerly Nocardia mediterranei or Streptomyces mediterranei). The crude antibiotic mixture was found to contain five closely related substances rifamycins A–E, but if the organism was cultured in the presence of sodium diethyl barbiturate (barbitone or barbital), the product was almost entirely rifamycin B (Figure 3.52). Rifamycin B has essentially no antibacterial activity, but may be converted chemically,

OH

OH

OH

H N

O OH OH

OH

O

OH

OH

O

OH

O

N

O

O H N

H N O

O O

O MeO

OH

N N

rifamycin SV

rifampicin

O OH

O O

O

O

CO2H

OH

H N

O

OH

H N

O O

rifamycin B

O O

NH N

O

OH

O

O

O

N

N N

N rifabutin

Figure 3.52

rifapentine

Me

The Acetate Pathway: Fatty Acids and Polyketides

89

Box 3.10 (continued) enzymically, or by biotransformation into rifamycin SV (Figure 3.52), a highly active antibacterial agent, and the first rifamycin to be used clinically. Further chemical modifications of rifamycin SV have produced better clinically useful drugs. Rifamycin SV is actually the immediate biosynthetic precursor of rifamycin B, and this conversion can be genetically blocked, resulting in the accumulation of rifamycin SV. Strain optimization has not been fruitful, however, and most commercial fermentations still rely on producing rifamycin B. The most widely used agent is the semi-synthetic derivative rifampicin (Figure 3.52). Rifampicin has a wide antibacterial spectrum, with high activity towards Gram-positive bacteria and a lower activity towards Gram-negative organisms. Its most valuable activity is towards Mycobacterium tuberculosis, and rifampicin is a key agent in the treatment of tuberculosis, usually in combination with at least one other drug to reduce the chances for development of resistant bacterial strains. It is also useful in control of meningococcal meningitis and leprosy. Rifampicin’s antibacterial activity arises from inhibition of RNA synthesis by binding to DNA-dependent RNA polymerase. RNA polymerase from mammalian cells does not contain the peptide sequence to which rifampicin binds, so RNA synthesis is not affected. In contrast to the natural rifamycins, which tend to have poor absorption properties, rifampicin is absorbed satisfactorily after oral administration and is also relatively free of toxic side-effects. The most serious side-effect is disturbance of liver function. A trivial, but to the patient potentially worrying, side-effect is discoloration of body fluids, including urine, saliva, sweat, and tears, to a red–orange colour, a consequence of the naphthalene/naphthoquinone chromophore in the rifamycins. Rifabutin (Figure 3.52) is a newly introduced derivative, which also has good activity against the Mycobacterium avium complex frequently encountered in patients with AIDS. Rifapentine (Figure 3.52) has a longer duration of action than the other anti-tubercular rifamycins.

(Figure 3.53). This simple structural analysis is essentially correct, though a novel feature here is that two proteins differing in their reducing nature are actually involved. Initial chain extension is carried out by a ‘reducing’ non-iterative type I PKS that constructs a C12 intermediate, which is then transferred to a second protein that lacks any KR, ER, and DH domains and carries out ‘non-reducing’ chain extension. The second protein possesses a starter unit-ACP transacylase (SAT) domain that loads the C12 chain for three further extensions.

Zearalenone (Figure 3.53), a toxin produced by the fungus Gibberella zeae and several species of Fusarium, has a relatively simple structure which is derived entirely from acetate/malonate units. It can be envisaged as a cyclization product from a polyketide where consecutive retention of several carbonyl functions will then allow formation of an aromatic ring by aldol condensation and enolizations near the carboxyl terminus – the processes of aromatic ring formation will be considered in more detail shortly (see page 99). Other parts of the chain have suffered considerable reductive modification

chain extension with reductions, etc

CO2H SCoA

SCoA

+ 5×

HO

EnzS

E1

O

O

O

O CO2H E2

OH

O O

aldol reaction, aromatization, lactone formation

O

SEnz O

HO

O HO

chain extension without reduction

O O zearalenone

Figure 3.53

O

SCoA

+ 3×

O

E1: ZEA2 E2: ZEA1

90 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition From a structural point of view, zearalenone is a remarkable example of an acetate-derived metabolite containing all types of oxidation level seen during the fatty acid extension cycle, i.e. carbonyl, secondary alcohol (eventually forming part of the lactone), alkene, and methylene, as well as having a portion which has cyclized to an aromatic ring because no reduction processes occur in that fragment of the chain. POLYKETIDE SYNTHASES: LINEAR POLYKETIDES AND POLYETHERS The macrolide systems described above are produced by formation of an intramolecular ester or amide linkage, utilizing appropriate functionalities in the growing polyketide chain. Macrolide formation does not always occur, and similar acetate/propionate precursors might also be expected to yield molecules which are essentially linear in nature. It is now possible to appreciate how some organisms, particularly marine microalgae, are able to synthesize polyunsaturated fatty acids such as EPA (Figure 3.54) directly by the use of PKS enzymes, instead of desaturating and elongating FAS-derived fatty acids (see page 49). The sequence proposed in the EPA-producing marine

O

OH

2 × malonyl-CoA

bacterium Shewanella pneumatophori allows chain extension with malonyl-CoA, introducing unsaturation via the KR/DH components. However, this would normally lead to a conjugated system of trans double bonds, as seen in the polyene antifungals (see page 82); the polyunsaturated fatty acids usually have a non-conjugated array of cis double bonds. EPA formation in Shewanella requires two additional enzyme activities, a 2,3-isomerase and a 2,2-isomerase, probably acting in concert with the DH (Figure 3.54). The first of these is an allylic isomerization (see page 51), whilst the second is a trans–cis isomerization, usually considered to require energy absorption (compare coumarins, page 161, and retinol, page 304). Discodermolide (Figure 3.55) does contain a lactone moiety, though this is in a six-membered ring. Nevertheless, the bulk of the molecule can be rationalized as a linear array of malonate- and methylmalonate-derived units. In common with the fatty acids, double bonds in the chain are also cis. This compound is synthesized by the deep-sea marine sponge Discodermia dissoluta, and is of considerable interest as the most potent natural promoter of tubulin assembly yet discovered. Taxol (see page 224) and the epothilones (see page 85) have a similar mode of action. It has also been

O

SCoA

O SEnz

SEnz

SEnz 2,3-I

DH

1. KS, KR, DH, ER 2. KS, KR

O

2,3-isomerase

KS, KR 2,2-isomerase

SEnz

OH

O SEnz

2,2-I

O

malonyl-CoA

SEnz

DH

1. KS, KR, DH, ER 2. KS, KR, DH/2,3-I

2 × malonyl-CoA

O

2 × malonyl-CoA

COSEnz

SEnz 1. KS, KR, DH/2,2-I 2. KS, KR, DH, ER 2 × malonyl-CoA O SEnz H

extender processing activities are indicated on arrows

1. KS, KR, DH/2,3-I 2. KS, KR, DH, ER CO2H

H

allylic isomerization (2,3-isomerase) eicosapentaenoic (EPA)

Figure 3.54

O

The Acetate Pathway: Fatty Acids and Polyketides OH

OH

OH

O OH

O

O

O

O

91

O

NH2

OH

via DEBS1 protein

discodermolide

Figure 3.55

monic acid

COSCoA HO 3-hydroxypropionate

9-hydroxynonanoic acid

+ malonyl-CoA

OH HO

O

O

OH

CO2H

O

O R

R = H, pseudomonic acid A R = OH, pseudomonic acid B

Met-derived methyls

decarboxylation −elimination

OH HO

PKS

SEnz OH

O

SEnz

SEnz

aldol

O O

O

O H

OH

SCoA

O allylic isomerization

acetyl-CoA O

ether ring formation, epoxidation, further hydroxylation pseudomonic acid A

HO

OR OH

OH

OH

esterification, reduction of double bond, hydroxylation of methyl

O

HO

SEnz OH

O

OH

Figure 3.56

found that taxol and discodermolide display synergistic action in combination. Discodermolide itself has proved rather too toxic for drug use, and studies have been restricted by limited supplies. It has been possible to prepare key intermediates for total synthesis by using genetically modified DEBS1 proteins (see page 71) expressed in a suitable host. Mupirocin is an antibiotic used clinically for the treatment of bacterial skin infections and for controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus (MRSA), when other antibiotics are ineffective. It has an uncommon mode of action, binding selectively to bacterial isoleucyl-tRNA synthase, which prevents incorporation of isoleucine into bacterial

proteins. Mupirocin is produced by Pseudomonas fluorescens and consists of a mixture of pseudomonic acids, the major components being pseudomonic acid A (about 90%) and pseudomonic acid B (about 8%) (Figure 3.56). These are esters of monic acids with a C9 saturated fatty acid, 9-hydroxynonanoic acid. The mupirocin gene cluster contains several domains resembling bacterial type I PKS and type I FAS systems, and other genes probably responsible for production of precursors and for later modifications. Parts of the sequence are particularly noteworthy, however. During PKS-mediated elaboration of the monic acid fragment, methyltransferase domains incorporate methyl groups, as seen with epothilone (page 86). A further

92 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition acid A formation in due course requires formation of an ether ring system and epoxidation of a double bond. Ether ring formation is thought to proceed through hydroxylation of one of the SAM-derived methyls. Molecules such as lasalocid A (Figure 3.57) from Streptomyces lasaliensis and monensin A (Figure 3.58) from Streptomyces cinnamonensis are representatives of a large group of compounds called polyether antibiotics. These and related compounds are of value in veterinary medicine, being effective in preventing and controlling coccidiae, and also having the ability to improve the efficiency of food conversion in ruminants. The polyether

methyl group originates from the methyl of an acetate precursor. Since the protein MupH bears considerable similarity to HMG-CoA synthase, the enzyme involved in mevalonate biosynthesis (see page 190), a similar aldol addition mechanism is proposed in this reaction (see also streptogramin A formation, Figure 7.27). Decarboxylation–elimination (again compare mevalonate biosynthesis) catalysed by MupH is then followed by an allylic elimination. This ‘methylation’ looks much more complicated than an SAM-mediated reaction, but is a rather neat way of inserting a methyl group onto a carbonyl, which is not possible with SAM. Pseudomonic

CO2H

epoxidations

O

HO OH

O

O

OH

OH O concerted cyclization sequence

O OH

lasalocid A

Figure 3.57

CO2H 5×

CO2H

CO2H SCoA

SCoA

+ 7×

O

SCoA

+

O

O

E1

HO O

hemiketal

OH

HO

O

HO OH

E1

EnzSOC

HO

O

O

HO2C

premonensin

epoxidations E2

concerted cyclization sequence

HO

HO OH

O

O

O O

HO

O

O

O

O O

E3 E4, E5 OMe

HO2C

O CO2H

E1: monensin PKS E2: MonC (epoxidase) E3: MonB (epoxide hydrolase)

Figure 3.58

E4: MonD (hydroxylase) E5: MonE (methyltransferase)

HO OH monensin A

The Acetate Pathway: Fatty Acids and Polyketides antibiotics are characterized by the presence of a number of tetrahydrofuran and/or tetrahydropyran rings along the basic chain. The polyether acts as an ionophore, increasing influx of sodium ions into the parasite, causing a resultant and fatal increase in osmotic pressure. Current thinking is that these ring systems arise via a cascade cyclization mechanism involving epoxide intermediates, as shown for lasalocid A (Figure 3.57). In the biosynthesis of monensin A (Figure 3.58), a modular PKS assembles a polyketide chain from malonate, methylmalonate, and ethylmalonate precursors to produce the triene shown, which is released as the hemiketal; the loading module contains a KSQ domain and the starter is malonate (see page 73). Following triepoxide formation, a concerted stereospecific cyclization sequence initiated by the hemiketal hydroxyl could proceed as indicated. Hydroxylation of the starter methyl group and methylation of a hydroxyl are late steps. Even more remarkable polyether structures are found in some toxins produced by marine dinoflagellates, which are in turn taken up by shellfish, thus passing on their toxicity to the shellfish. Okadaic acid (Figure 3.59) and related polyether structures from Dinophysis species are responsible for diarrhoeic shellfish poisoning in mussels, causing severe diarrhoea, nausea, and vomiting in consumers of contaminated shellfish in many parts of the world. Okadaic acid is now known to be a potent inhibitor of protein phosphatases, and causes dramatic increase in the phosphorylation of many proteins, leading to the observed cellular responses. It has become a useful pharmacological tool in the study of protein phosphorylation processes. Brevetoxin A and brevetoxin B (Figure 3.60) are examples of the toxins associated with ‘red tide’ blooms of dinoflagellates which affect fishing and also tourism, especially in Florida and the Gulf of Mexico. The red tide toxins are derived from Karenia brevis (formerly Gymnodimium breve) and are the causative agents of neurotoxic shellfish poisoning, leading to neurological disorders and gastrointestinal troubles. The toxins are known to bind to sodium channels, keeping them in an

H

HO2C OH

open state. Fatalities among marine life, e.g. fish, dolphins, and whales, and in humans are associated with these toxins synthesized by organisms at the base of the marine food chain. These compounds are postulated to be produced from a polyunsaturated fatty acid by epoxidation of double bonds, and then a concerted sequence of epoxide ring openings leading to the extended polyether structure of trans-fused rings (Figure 3.60). The carbon skeletons of okadaic acid and the brevetoxins do not seem to arise from acetate/propionate units in the systematic manner as seen with monensin A; biosynthetic studies have shown that fragments from the citric acid cycle and other precursors become involved. This may reflect some significant differences between terrestrial and marine microorganisms. Ciguatoxin (Figure 3.60) is one of the more complex examples of a polyether structure found in nature, though it is certainly not the largest. This compound is found in the moray eel (Gymnothorax javanicus) and in a variety of coral reef fish, such as red snapper (Lutjanus bohar), though some 100 different species of fish may cause food poisoning (ciguatera) in tropical and subtropical regions. Ciguatoxin is remarkably toxic even at microgram levels; ciguatera is characterized by vomiting, diarrhoea, and neurological problems. Most sufferers slowly recover, though this may take a considerable time, and few cases are fatal due principally to the very low levels of toxin actually present in the fish. The dinoflagellate Gambierdiscus toxicus is ultimately responsible for polyether production, synthesizing a less toxic analogue which is passed through the food chain and eventually modified into the very toxic ciguatoxin by the fish. More than 20 ciguatoxin-like structures have been identified. Halichondrins are both polyethers and macrolides, and were originally isolated from the Pacific marine sponge Halichondria okadai. They have received considerable attention because of their complex structures and their extraordinary antitumour activity. Halichondrin B (Figure 3.61) was shown to affect tubulin polymerization, though at a site distinct from the Catharanthus alkaloid site (see page 375). To

O

O O H OH

H

H

OH O

O H

okadaic acid

Figure 3.59

93

O

H

OH

H

O

94 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition HO CHO

EnzS H

HO

O

CHO

epoxidations O O O O

O

O

O

O

O

O O

HO H

concerted cyclization sequence

O

H

H

H

H O

OH H

H

H

O H O

H

O

O H H

O

O

H H

H

brevetoxin A

H

O

O

H

H

O

O H H

O H

O

H O

O

H O H HO

O

H

CHO

HO

O

H

CHO

H O

H

H

H

O

H brevetoxin B H

HO

H

O H

H HO

OH

O

O H

H H O HO H

H

O

H H O

H

H

O

O H OH H

O

O H H

H H H O

O O

H

H OH

ciguatoxin

Figure 3.60 facilitate clinical studies, halichondrins were produced by total synthesis, leading to the discovery that it was only necessary to have the macrocyclic portion to maintain the biological activity; the macrocyle still contains several complex ether linkages, however. Substitution of a ketone for the lactone function increased

stability; in due course, the modified derivative eribulin (Figure 3.61) has proved successful in the treatment of advanced breast cancer and has been introduced as a drug. The zaragozic acids (squalestatins) are structurally rather different from the polyethers discussed so far, but

The Acetate Pathway: Fatty Acids and Polyketides lactone H H HO H

H O

O

H

H

O

HO

O O

O H

H

H

O

H

H

ketone H2N

H

O O

O

H

H

O

95

O

OH

OH

OMe H

H

H O

O H

H O

O

O

O

H

H O

H O

H

O

O

O

O

O

halichondrin B

eribulin

Figure 3.61

acetate/malonate + methionine

E1: squalestatin tetraketide synthase (SQTKS)

OH E1

PKS domain organization

O

KS ⎯ AT ⎯ DH ⎯ CMeT ⎯ ER ⎯ KR ⎯ ACP

CMeT: C-methyl transferase OH

O

O

OAc H3C

O HO2C HO2C

COSCoA

• Met

CoAS

O OH

HO2C

CO2H succinic acid

O CO2H

H

zaragozic acid A (squalestatin S1)

Figure 3.62

they are primarily acetate derived, and the central ring system is suggested to be formed by an epoxide-initiated process resembling the polyether derivatives just described. Thus, zaragozic acid A (squalestatin S1; Figure 3.62) is known to be constructed from two acetate-derived chains and a C4 unit such as the Krebs cycle intermediate oxaloacetate (succinic acid can act as precursor). One chain has a benzoyl-CoA starter (from the shikimate pathway, see page 157), and both contain two methionine-derived side-chain substituents (Figure 3.62). An iterative (repeating) type I PKS catalysing synthesis of one of these has been characterized. In common with a number of fungal PKSs, a C-methyl transferase domain is included in the protein, and C-methylation is part of the PKS activity (see also epothilones, page 86, and mupirocin, page 91). Iterative PKSs do not have the modular system to control the order and composition of extender units added.

Instead, programming of the sequence is encoded in the PKS itself, in a manner that remains to be clarified. The heterocyclic ring system can be envisaged as arising via nucleophilic attack onto oxaloacetic acid, formation of a diepoxide, then a concerted sequence of reactions as indicated (Figure 3.63). The zaragozic acids are produced by a number of fungi, including Sporomiella intermedia and Leptodontium elatius, and are of considerable interest since they are capable of reducing blood cholesterol levels in animals by acting as potent inhibitors of the enzyme squalene synthase (see page 235). This is achieved by mimicking the steroid precursor presqualene PP and irreversibly inactivating the enzyme; both compounds have lipophilic side-chains flanking a highly polar core. They have considerable medicinal potential for reducing the incidence of coronary-related deaths (compare the statins, below).

96 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition aldol reaction

O HO2C

HO2C HO

HO2C H

O

concerted nucleophilic reactions

H O

HO2C O

formation of epoxide functions

O

O

HO2C

OO

HO2C CO2H H2O

CO2H

CO2H

HO

HO2C HO2C HO

O O CO2H

oxaloacetic acid

Figure 3.63 DIELS–ALDER CYCLIZATIONS A number of cyclic structures, typically containing cyclohexane rings, are known to be formed via the acetate pathway and can be rationalized as involving an enzymic Diels–Alder reaction (Figure 3.64), though only one example will be considered here. Thus, lovastatin arises from a C18 polyketide chain with C-methylation, and relatively few of the oxygen functions are retained in the final product. A secondary polyketide chain, with methylation, provides the ester side-chain (Figure 3.65). A Diels–Alder reaction accounts for formation of the decalin system as shown, and this is now known to occur at the hexaketide intermediate stage, catalysed by the PKS itself, though it is not known just how this is accomplished. The PKS (lovastatin nonaketide synthase) is an iterative type I system and carries a C-methyltransferase domain. Unusually, though the PKS carries an ER domain, this function is not activated unless an accessory protein LovC is also present. The product of lovastatin nonaketide synthase is the lactone dihydromonacolin L, and this is subjected to a sequence of post-PKS modifications, leading to monacolin J. The ester side-chain is produced by a non-iterative type I PKS, lovastatin diketide synthase, which also carries a C-methyltransferase domain. It carries no TE domain, and the fatty acyl chain appears to be delivered directly from the PKS protein in conjunction with the esterifying enzyme.

diene

dienophile Diels–Alder reaction

Figure 3.64

The C-methylation processes, quite common with fungal PKS systems, are easily interpreted as in Figure 3.66. The ketoester intermediate may be methylated with SAM via an enolate anion mechanism; the product then re-enters the standard chain extension process. Lovastatin was isolated from cultures of Aspergillus terreus and was found to be a potent inhibitor of hydroxymethylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme in the mevalonate pathway (see page 189). Analogues of lovastatin (statins) find drug use as HMG-CoA reductase inhibitors, thus lowering blood cholesterol levels in patients [Box 3.11]. These drugs have rapidly become the top-selling drugs worldwide. POLYKETIDE SYNTHASES: AROMATICS In the absence of any reduction processes, the growing poly-β-keto chain needs to be stabilized on the enzyme surface until the chain length is appropriate, at which point cyclization or other reactions can occur. A poly-β-keto ester is very reactive, and there are various possibilities for undergoing intramolecular Claisen or aldol reactions, dictated of course by the nature of the enzyme and how the substrate is folded. Methylenes flanked by two carbonyls are activated, allowing formation of carbanions/enolates and subsequent reaction with ketone or ester carbonyl groups, with a natural tendency to form strain-free six-membered rings. Aromatic compounds are typical products from type II and type III PKSs, though there are a few examples where type I PKSs produce aromatic rings. Whilst the chemical aspects of polyketide synthesis are common to all systems, a very significant difference compared with most of the type I systems described under macrolides is that the processes are iterative rather than non-iterative. In a non-iterative system, the sequence of events is readily related to the sequence of enzymic domains as each is brought into play. It is less

The Acetate Pathway: Fatty Acids and Polyketides

SCoA O

KR DH

KR DH

KR DH CMeT

EnzS

CO2H

SEnz EnzS

O

ER O

SCoA

KR DH

ER

ER

O

O

HO

O

KR DH

HO

O

Diels–Alder cyclase

COSEnz OH

O

O H

H

KR

KR

KR DH ER

extender processing activites are indicated on arrows

PKS domain organization

LovB: KS ⎯ AT ⎯ DH ⎯ CMeT ⎯ (ER) ⎯ KR ⎯ ACP LovC:

E1 + E2 hydroxylation

O2 NADPH

H

H

H dihydromonacolin L

SEnz

H

ER

CMeT: C-methyl transferase

E4

HO

O

HO

O

O

HO

O

O 1,4-elimination

H

O hydroxylation

H

− H2O

O2 NADPH

HO

H

OH α-hydroxy-3,5-dihydromonocolin L SCoA O

O

CMeT,KR DH, ER

O SEnz

CO2H

monocolin L

monocolin J O

HO

SCoA O E3 E1: E2: E3: E4: E5:

PKS domain organization LovF: KS ⎯ AT ⎯ DH ⎯ CMeT ⎯ ER ⎯ KR ⎯ ACP

lovastatin nonaketide synthase (LNKS; LovB) LovC (accessory protein) lovastatin diketide synthase (LDKS; LovF) LovA LovD

Figure 3.65

esterification

E5 SEnz

O O

O O

H

lovastatin

97

98 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O RCH2

O SEnz

OH

RCH2

ER

SEnz

standard reduction processes

KS

O

RCH2

SEnz

CMeT

O

O

RCH2

O RCH2

ER

DH

SEnz Ad S

H3C KR OH

SEnz

RCH2 SEnz

alkylation at ketoester stage precedes reduction

O

O

O

SAM

KS

SEnz

SEnz

KR

O

RCH2

RCH2

DH

O

SAM C-alkylation with SAM

O

RCH2

SEnz

Figure 3.66

Box 3.11 Statins Mevastatin (formerly compactin; Figure 3.67) is produced by cultures of Penicillium citrinum and Penicillium brevicompactum, and was shown to be a reversible competitive inhibitor of HMG-CoA reductase, dramatically lowering sterol biosynthesis in mammalian cell cultures and animals, and reducing total and low-density lipoprotein (LDL) cholesterol levels (see page 251). Mevastatin in its ring-opened form (Figure 3.68) mimics the half-reduced substrate mevaldate hemithioacetal during the two-stage reduction of HMG-CoA to mevalonate (see page 190), and the affinity of this agent towards HMG-CoA reductase is some 104 -fold greater than that of the normal substrate. High blood cholesterol levels contribute to the incidence of coronary heart disease, so mevastatin and analogues are of potential value in treating high-risk coronary patients, and several agents are already in use. These are generically known as statins, and they have rapidly become the top-selling drugs worldwide. As the liver synthesizes less cholesterol, this in turn stimulates the production of high-affinity LDL receptors on the surface of liver cells. Consequently, the liver removes more LDL from the blood, leading to the reduction of blood levels of both LDL and cholesterol. The clinical use of statins has also revealed that these drugs promote beneficial effects on cardiovascular functions that do not correlate simply with their ability to lower cholesterol levels. Statins are now routinely prescribed to slow the progression of coronary artery disease and to reduce mortality from cardiovascular disease. They have been suggested to have potentially useful anti-inflammatory and anticancer activities, and some are also being tested against Alzheimer’s disease and osteoporosis. Lovastatin (formerly called mevinolin or monacolin K; Figure 3.67) is produced by Monascus ruber and Aspergillus terreus and is slightly more active than mevastatin. It was the first statin to be marketed, but has since been superseded by more active

HO O

O O

H

HO

O

O O

O O

H

HO

O

HO

O

O O

O O

H

H

HO mevastatin

Figure 3.67

lovastatin (mevinolin; monacolin K)

simvastatin

CO2H OH

pravastatin

The Acetate Pathway: Fatty Acids and Polyketides

99

Box 3.11 (continued) HO

HO

CO2H

HO

CO2H

CO2H

OH

OH F

OH F

F N

N N NH O

MeO2S

N N

Me

rosuvastatin

fluvastatin atorvastatin

Figure 3.67 (continued) HO

HO

CO2H

CO2H O

OH

O

H O

H

H

H

NADPH

HO

CO2H

CO2H O

OH

E1

CoAS

CoAS

HMG-CoA

HO

H

mevaldic acid E1

mevaldic acid hemithioacetal HO

E1: HMG-CoA reductase

NADPH CO2H OH

mevastatin (opened lactone form)

mevalonic acid

Figure 3.68 agents. Simvastatin is obtained fron lovastatin by ester hydrolysis and then re-esterification, and is two to three times as potent as lovastatin. Pravastatin is prepared from mevastatin by microbiological hydroxylation using Streptomyces carbophilus and is consequently more hydrophilic than the other drugs, with an activity similar to lovastatin. Lovastatin and simvastatin are both lactones, but at physiological pHs they exist in equilibrium with the open-ring hydroxy acids; only the open-ring form is biologically active. Other agents currently in use are synthetic, though they feature the same 3,5-dihydroxycarboxylic acid side-chain as in pravastatin. Atorvastatin, fluvastatin, and rosuvastatin have all been introduced recently, and others are in development.

clear how a repeating process can be closely programmed to produce the final product, and this is somehow encoded in the PKS itself. Thus, the mechanisms of Figure 3.69 will need to be repeated by reuse of the same domains. Type II systems do contain two KS domains (KSα and KSβ ), though only the KSα domain is implicated in the Claisen condensation. KSβ lacks the active site cysteine and appears analogous to the KSQ of some macrolide PKSs (see page 73), so it may be involved in loading and decarboxylation of malonyl-CoA. Additional domains for KR, cyclase, or aromatase activities may be present. Type III systems differ from the others in the use of coenzyme

A esters rather than ACPs (Figure 3.69). The PKS is a single protein, though it exists as a homodimer in its native form. It is not appropriate here to attempt subdivisions according to the PKS type, and compounds will be considered primarily according to structural features.

Cyclizations The poly-β-keto ester (Figure 3.70), formed from four acetate units (one acetate starter group and three malonate chain extension units), is capable of being folded in at least two ways, A and B (Figure 3.70). For A, ionization

100 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition type II PKS (iterative) KSα KSβ

KSα KSβ

ACP

SH

SH

KSα KSβ

ACP

S

S

additional subunits: ketoreductases, cyclases, aromatases

S

SH O

O

O

ACP

R O

O

R

O H type III PKS (iterative; ACP-independent) load

condensation

Enz

transfer

Enz

SH

Enz

Enz

SCoA

S

SH

O

O

S

SCoA O

O

R O

O

O O H

R

R

Figure 3.69

CO2H SCoA

SCoA +

SEnz

3×

O

O

acetyl-CoA

O

malonyl-CoA

O

O

O

poly-β-keto ester folding A

aldol reaction

OH

O

folding B O

O O

O

SEnz

O

O

O

O O

O

enolization hydrolysis

O

OH

HO

Figure 3.70

O

O

O

enolization CO2H OH

enolization favoured by formation of aromatic ring

SEnz

re-formation of carbonyl possible by expulsion of leaving group; this also releases product from enzyme

SEnz O

O

SEnz

dehydration favoured by formation of conjugated system

O

O

Claisen reaction

H

SEnz H

O

O

orsellinic acid

HO

OH

phloracetophenone

O enolization favoured by formation of aromatic ring

O

The Acetate Pathway: Fatty Acids and Polyketides 101 SEnz

acetyl-CoA malonyl-CoA

E1

O

O

E1: 6-methylsalicylic acid synthase PKS domain organization

O

KS ⎯ AT ⎯ DH ⎯ KR ⎯ ACP

enolization then ester formation

NADPH HO

O

reduction of carbonyl to alcohol in presence of NADPH

HO

O SEnz

O SEnz

O

O

E1

O triacetic acid lactone

O – H 2O malonyl-CoA O

O SEnz O

enolization hydrolysis

aldol reaction – H2 O

SEnz

SEnz chain extension after dehydration / removal O of oxygen function

O

O

O

E1

CO2H OH 6-methylsalicylic acid

Figure 3.71 of the α-methylene allows aldol addition onto the carbonyl six carbon atoms distant along the chain, giving the tertiary alcohol. Dehydration occurs as in most chemical aldol reactions, giving the alkene, and enolization follows to attain the stability conferred by the aromatic ring. The enzyme–thioester linkage is then hydrolysed to produce orsellinic acid. Alternatively, folding of the polyketo ester as in B allows a Claisen reaction to occur, which, although mechanistically analogous to the aldol reaction, is terminated by expulsion of the thiol leaving group and direct release from the enzyme. Enolization of the cyclohexatrione produces phloracetophenone. As with other PKSs, the whole sequence of reactions is carried out by an enzyme complex which converts acetyl-CoA and malonyl-CoA into the final product without giving any detectable free intermediates, thus combining PKS and polyketide cyclase activities. A distinctive feature of an aromatic ring system derived through the acetate pathway is that several of the carbonyl oxygens of the poly-β-keto system are retained in the final product. These end up on alternate carbon atoms around the ring system. Of course, one or more might be used in forming a carbon–carbon bond, as in orsellinic acid. Nevertheless, this oxygenation on alternate carbon atoms, a meta oxygenation pattern, is usually easily recognizable and points to the biosynthetic origin of the molecule. This meta oxygenation pattern contrasts with that seen on aromatic rings formed via the shikimate pathway (see page 140).

6-Methylsalicylic acid (Figure 3.71) is a metabolite of Penicillium patulum, and differs from orsellinic acid by the absence of a phenol group at position 4. It is also derived from acetyl-CoA and three molecules of malonyl-CoA, and the ‘missing’ oxygen function is removed during the biosynthesis. Orsellinic acid is not itself deoxygenated to 6-methylsalicylic acid. The enzyme 6-methylsalicylic acid synthase is one of the smallest PKSs known and is of the iterative type I group. It requires NADPH as cofactor and removes the oxygen function by employing its KR domain just once. Reduction of the ketone to an alcohol, followed by a dehydration step, occurs on a six-carbon intermediate after the second chain extension (Figure 3.71). Further chain extension is then followed by aldol condensation, enolization, and release from the enzyme to generate 6-methylsalicylic acid. Important evidence for reduction occurring at the C6 stage comes from the formation of triacetic acid lactone (Figure 3.71) as a ‘derailment product’ if NADPH is omitted from the enzymic incubation. The PKS is iterative, the enzyme activities acting repeatedly, but there is some programming that allows KR to participate only after the second extension round. In the absence of NADPH, KR remains inactive. There is no TE domain; perhaps release from the enzyme is not simple hydrolysis. The folding of a polyketide chain can be established by labelling studies, feeding carbon-labelled sodium acetate to the appropriate organism and establishing the position of labelling in the final product by chemical degradation and counting (for the radioactive isotope 14 C)

102 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition and lactone or ester functions are thus reasonably common. For example, the lichen metabolite lecanoric acid is a depside (an ester formed from two phenolic acids) and the result from combination of two orsellinic acid thioester molecules (Figure 3.73). The lactone ring in 6-hydroxymellein (Figure 3.74) is formed by reaction of the end-of-chain thioester with a hydroxyl function in the starter group. The original keto group is reduced during chain extension, at the C6 stage, as noted in the formation of 6-methylsalicylic acid. The same derailment metabolite, triacetic acid lactone, is produced by the enzyme in the absence of NADPH. Unusually, the carbonyl group reduced is that of the starter unit rather than of the extender. SAM-dependent methylation of one of the hydroxyls leads to 6-methoxymellein, an antifungal stress metabolite of carrot (Daucus carota; Umbelliferae/Apiaceae). 6-Hydroxymellein synthase is a homodimeric protein, and may well be a type III plant PKS; type III systems utilize coenzyme A esters rather than ACPs.

or by NMR spectrometry (for the stable isotope 13 C). 13 C NMR spectrometry is also valuable in establishing the location of intact C2 units derived from feeding 13 C2 -labelled acetate (see page 36). This is exemplified in Figure 3.72, where alternariol, a metabolite from the mould Alternaria tenuis, can be established to be derived from a single C14 polyketide chain, folded as shown, and then cyclized. Whilst the precise sequence of reactions involved is not known, paper chemistry allows us to formulate the essential features. Two aldol condensations followed by enolization in both rings would give a biphenyl, and lactonization would then lead to alternariol. The oxygenation pattern in alternariol shows alternate oxygen atoms on both aromatic rings, and an acetate origin is readily surmised, even though some oxygen atoms have been used in ring formation processes. The lone methyl ‘start-of-chain’ is also usually very obvious in acetate-derived compounds, though the carboxyl ‘end-of-chain’ can often react with convenient hydroxyl functions, which may have arisen through enolization,

OH

O

*

* 7 × H3C

*

* CO2H

* O

*

O

O

* OH

O

*

*

* OO

*

HO

SEnz

* O

O

alternariol

C14 poly-β-keto chain refers to intact acetate C2 unit

*

*

*

2 × aldol – H2O

lactone formation

O

OH enolization O

O

O

OH

COSEnz

COSEnz OH

HO

Figure 3.72

OH

OH

COSEnz

SEnz HO

HO

OH orsellinic acid (as thioester)

Figure 3.73

CO2H

O

O

O HO

OH lecanoric acid

The Acetate Pathway: Fatty Acids and Polyketides 103 acetyl-CoA / malonyl-CoA

O

E1 malonyl-CoA

O

O

NADPH

O

EnzS

O

O

OH

EnzS E1

O

O

SEnz

malonyl-CoA SEnz OH O

O

O

OH

O E1

HO OH

O O triacetic acid lactone E1: 6-hydroxymellein synthase E2: 6-hydroxymellein O-methyltransferase

O

OH O

SAM E2

MeO 6-methoxymellein

O O

HO 6-hydroxymellein

Figure 3.74 Post-Polyketide Synthase Modifications A number of natural anthraquinone derivatives are also excellent examples of acetate-derived structures. Structural analysis of endocrocin (Figure 3.75) found in species of Penicillium and Aspergillus fungi suggests folding of a polyketide containing eight C2 units to form the periphery of the carbon skeleton. Three aldol-type condensations would give a hypothetical intermediate 1, and, except for a crucial carbonyl oxygen in the centre ring, endocrocin results by enolization reactions, one of which involves the vinylogous enolization —CH2 —CH CH—CO— → —CH CH—CH C(OH)—. The additional carbonyl oxygen must be introduced at some stage during the biosynthesis by an oxidative process, for which we have little information. Emodin, a metabolite of some Penicillium species, but also found in higher plants, e.g. Rhamnus and Rumex species, would appear to be formed from endocrocin by a simple decarboxylation reaction. This is facilitated by the adjacent phenol function (see page 22). O-Methylation of emodin would then lead to physcion. Islandicin is another anthraquinone pigment produced by Penicillium islandicum, and differs from emodin in two ways: one hydroxyl is missing and a new hydroxyl has been incorporated adjacent to the methyl. Without any evidence for the sequence of such reactions, the structure of hypotheical intermediate 2 shows the result of three aldol condensations with reduction of a carbonyl, though the latter would most likely occur during chain extension. A dehydration reaction, two oxidations, and a decarboxylation are necessary to attain the islandicin structure. In chrysophanol, aloe-emodin and rhein, the same oxygen function is lost by reduction as in islandicin, and decarboxylation

also occurs. The three compounds are interrelated by a sequential oxidation of the methyl in chrysophanol to a hydroxymethyl in aloe-emodin and a carboxyl in rhein. As noted in the case of macrolides, structural modifications undergone by the basic polyketide are also conveniently considered according to the timing of the steps in the synthetic sequence. Thus, ‘missing’ oxygen functions appear to be reduced out well before the folded and cyclized polyketide is detached from the enzyme, and are mediated by the KR component of the PKS enzyme complex. On the other hand, reactions like decarboxylation, O-methylation, and sequential oxidation of a methyl to a carboxyl are representative of post-PKS transformations occurring after the cyclization reaction. These later conversions can often be demonstrated by the isolation of separate enzymes catalysing the individual steps. Most of the secondary transformations are easily rationalized by careful consideration of the reactivity conferred on the molecule by the alternating and usually phenolic oxygenation pattern. These oxygen atoms activate adjacent sites, creating nucleophilic centres. Introduction of additional hydroxyl groups ortho or para to an existing phenol will be facilitated (see page 26), allowing the extra hydroxyl of islandicin to be inserted, for example. The ortho- or para-diphenols are themselves susceptible to further oxidation in certain circumstances, and may give rise to ortho- and para-quinones (see page 26). The quinone system in anthraquinones is built up by an oxidation of the central cyclohexadienone ring, again at a nucleophilic centre activated by the enone system. Methyl groups on an aromatic ring are also activated towards oxidation, facilitating the chrysophanol → aloe-emodin oxidation, for example. Decarboxylation, e.g. endocrocin → emodin,

104 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O O aldol reactions – H 2O

O

O

O O

O O

SEnz

hypothetical poly-β-keto ester (no reduction)

O

NADPH

– H 2O

– H2O

O

HO

O

NADPH reduces carbonyl

NADPH

O

O

HO CO2H

CO2H

CO2H

O O O hypothetical intermediate 2

O O O hypothetical intermediate 1 enolizations oxidation O

O

O O O hypothetical intermediate 2

–H2O enolization oxidation – CO2

–H2O enolization 2 × oxidation – CO2

O

OH

HO CO2H OH O OH endocrocin

OH O OH chrysophanol

decarboxylation facilitated by ortho-hydroxyl

– CO2

O

MeO SAM

HO

OH

O OH emodin

O-methylation of phenol

O OH islandicin

oxidation of methyl to alcohol

O O

O

OH

O CO2H

OH O

OH O OH physcion

OH O OH aloe-emodin

oxidation of alcohol to acid OH

O rhein

OH

Figure 3.75 is readily achieved in the presence of an ortho phenol function, though a para phenol can also facilitate this (see page 22). It is now appreciated that the assembly of the anthraquinone skeleton (and related polycyclic structures) is achieved in a stepwise sequence. After the polyketide chain is folded, the ring at the centre of the fold is formed first, followed in turn by the next two rings. The pathway outlined for the biosynthesis of endocrocin and emodin is shown in Figure 3.76. Mechanistically, there is little difference between this and the speculative pathway of Figure 3.75, but the sequence of reactions is altered. Decarboxylation appears to take place before aromatization of the last-formed ring system, and tetrahydroanthracene intermediates, such as atrochrysone carboxylic acid and atrochrysone, are involved. These dehydrate to the anthrones endocrocin anthrone and emodin anthrone respectively prior to introduction of the extra carbonyl

oxygen as a last transformation in the production of anthraquinones. This oxygen is derived from O2 . The interrelationships of Figure 3.76 are derived mainly from feeding experiments in fungi. A type III octaketide synthase gene from the anthraquinone-producing plant Aloe arborescens, when expressed in Escherichia coli, did not synthesize anthraquinones, but instead the two products SEK 4 and SEK 4b, not normally found in Aloe (Figure 3.77). These obviously result from different folding arrangements of the polyketide precursor. Thus, the expressed protein seems to lack some agent to fold the chain correctly or perhaps a cyclase element; the observed products may result from non-enzymic cyclization reactions. This protein utilizes just malonyl-CoA, so the starter unit is generated by decarboxylation (see page 73). Emodin, physcion, chrysophanol, aloe-emodin, and rhein form the basis of a range of purgative anthraquinone

The Acetate Pathway: Fatty Acids and Polyketides 105 O

HO O O

O O

O O

aldol, – H2O enolization

O

SEnz O

O

O O

O O

SEnz O

– H2O aldol, – H2O enolization

E1

HO O OH

O

O O

O

SEnz OH

O

O

O O

SEnz O

aldol, – H2O enolization

O

HO O

O OH

OH

SEnz

O

O

OH O OH chrysophanol anthrone

OH O OH chrysophanol O

aldol

O

O

OH

HO

SEnz OH

OH

O

O

HO – H2O

O

CO2H OH

O rhein

OH

O

OH

OH

OH

OH O OH aloe-emodin

hydrolysis from enzyme HO

CO2H

OH O

O

CO2H OH

atrochrysone carboxylic acid

HO

O

OH

CO2H OH O OH endocrocin

endocrocin anthrone

– CO2 OH

HO

OH

OH

O HO – H2O

O2

E1

E2

O

atrochrysone E1: emodin anthrone synthase E2: emodin anthrone oxygenase

Figure 3.76

O

HO

OH

O

OH

emodin anthrone

MeO SAM

OH

O OH emodin

OH O OH physcion

106 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O OH O

O O

O O E1 HO

≡

O

O

O

O O

O O

O

SEnz O

O

O

≡

E1 OH OO

O

O O SEnz O

O

O SEnz

O HO

HO

O

O

O

O

HO

O

O SEK 4b

SEK 4

E1: octaketide synthase

Figure 3.77

OH

O

OH

OH

O

OH

– H+, – 1e

radical coupling

O-glycosylation OH

O

OH

CO2H

CO2H tautomerism OH

OH

OH

CO2H UDPGlc

CO2H

CO2H

CO2H

resonance OH

O

OH

OH

– H+, – 1e CO2H

O

×2

rhein anthrone

OH

GlcO

O

OH

rhein dianthrone (sennidin A/B)

GlcO

O

OH

sennoside A/B

CO2H

rhein anthranol

Figure 3.78 derivatives found in long-established laxatives such as senna [Box 3.12]. The free anthraquinones themselves have little therapeutic activity and need to be in the form of water-soluble glycosides to exert their action. Although simple anthraquinone O-glycosides are present in the drugs, the major purgative action arises from compounds such as sennosides, e.g. sennosides A and B (Figure 3.78), which are dianthrone O-glycosides. These types of derivative are likely to be produced from interme-

diate anthrone structures. A one-electron oxidation allows oxidative coupling (see page 28) of two anthrone systems to give a dianthrone (Figure 3.78). This can be formulated as direct oxidation at the benzylic –CH2 –, or via the anthranol, which is the phenolic tautomer of the anthrone. Glycosylation of the dianthrone system would then give a sennoside-like product; alternatively, glycosylation may precede the coupling.

Box 3.12 Senna Senna leaf and fruit are obtained from Cassia angustifolia (Leguminosae/Fabaceae), known as Tinnevelly senna, or less commonly from Cassia senna (syn Cassia acutifolia), which is described as Alexandrian senna. The plants are low, branching shrubs, Cassia angustifolia being cultivated in India and Pakistan, and Cassia senna being produced in the Sudan, much of it from wild plants. Early harvests provide leaf material, whilst both leaf and fruit (senna pods) are obtained later on. There are no significant differences in the chemical constituents of the two sennas, or between leaf and fruit drug. However, amounts of the active constituents do vary, and this appears to be a consequence of cultivation conditions and the time of harvesting of the plant material.

The Acetate Pathway: Fatty Acids and Polyketides 107

Box 3.12 (continued) RO

O

OH

R H

O

H

CO2H

OH

R = Glc, sennoside A R = H, sennidin A

O

OH

R

CO2H

H R

RO

RO

O

H

CO2H

OH

R = Glc, sennoside B R = H, sennidin B

O

OH

R

CO2H

H S

RO

RO

RO

RO

O

H

OH

R

OH

R = Glc, sennoside C R = H, sennidin C

OH

R

CO2H

H

O

CO2H

H

OH

S

RO

O

OH

R = Glc, sennoside D R = H, sennidin D

Figure 3.79 The active constituents in both senna leaf and fruit are dianthrone glycosides, principally sennosides A and B (Figure 3.79). These compounds are both di-O-glucosides of rhein dianthrone (sennidins A and B) and liberate these aglycones on acid hydrolysis; oxidative hydrolysis (e.g. aqueous HNO3 or H2 O2 /HCl) produces the anthraquinone rhein (Figure 3.76). Sennidins A and B are optical isomers: sennidin A is dextrorotatory (+) whilst sennidin B is the optically inactive meso form. Minor constituents include sennosides C and D (Figure 3.79), which are also a pair of optical isomers, di-O-glucosides of heterodianthrones sennidins C and D. Sennidin C is dextrorotatory, whilst sennidin D is optically inactive, approximating to a meso form in that the modest change in substituent does not noticeably affect the optical rotation. Oxidative hydrolysis of sennosides C and D would produce the anthraquinones rhein and aloe-emodin (Figure 3.76). Traces of other anthraquinone glycoside derivatives are also present in the plant material. Much of the sennoside content of the dried leaf appears to be formed by enzymic oxidation of anthrone glycosides during the drying process. Fresh leaves and fruits also seem to contain primary glycosides which are more potent than sennosides A and B, and which appear to be partially hydrolysed to sennosides A and B (the secondary glycosides) by enzymic activity during collection and drying. The primary glycosides contain additional glucose residues. Senna leaf suitable for medicinal use should contain not less than 2.5% dianthrone glycosides calculated in terms of sennoside B. The sennoside content of Tinnevelly fruits is between 1.2 and 2.5%, with that of Alexandrian fruits being 2.5–4.5%. Senna preparations, in the form of powdered leaf, powdered fruit, or extracts, are typically standardized to a given sennoside content. Non-standardized preparations have unpredictable action and should be avoided. Senna is a stimulant laxative and acts on the wall of the large intestine to increase peristaltic movement. After oral administration, the sennosides are transformed by intestinal flora into rhein anthrone (Figure 3.78), which appears to be the ultimate purgative principle. The glycoside residues in the active constituents are necessary for water solubility and subsequent transportation to the site of action. Although the aglycones, including anthraquinones, do provide purgative action, these materials are conjugated and excreted in the urine after oral administration rather than being transported to the colon. Senna is a purgative drug suitable for either habitual constipation or for occasional use, and is widely prescribed.

However, further oxidative steps on a dianthrone, e.g. emodin dianthrone (Figure 3.80), can create a dehydrodianthrone and then allow oxidative coupling of the aromatic rings through protohypericin to give a naphthodianthrone, in this case hypericin. The reactions of Figure 3.80 can also be achieved chemically by passing air into an alkaline solution of emodin anthrone, though yields are low. Hypericin is found in cultures of Dermocybe fungi and is also a constituent of St John’s Wort, Hypericum perforatum (Guttiferae/Hypericaceae). A protein obtained by expression of a Hypericum perforatum gene in Escherichia coli has been observed to transform

the anthraquinone emodin into hypericin, presumably by a sequence similar to that shown. However, since emodin rather than emodin anthrone was the substrate, some additional step must be involved. An aldol condensation between emodin and emodin anthrone could provide the dehydrodianthrone as an alternative to the oxidative coupling. Hypericum perforatum is a popular herbal medicine in the treatment of depression [Box 3.13]. The naphthodianthrones have no purgative action, but hypericin can act as a photosensitizing agent in a similar manner to furocoumarins (see page 165). Thus, ingestion of hypericin results in an increased absorption of UV light and can

108 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH

O

OH

radical coupling

OH

O

OH

×2

HO

HO emodin anthrone OH

O

HO

OH HO OH O OH emodin dianthrone

+ HO OH O OH emodin anthrone

O emodin

aldol reaction

O

E1 OH

O

OH

OH

O

HO HO

radical coupling OH O OH hypericin

O

OH

O

HO HO

radical coupling OH O OH protohypericin

OH

O

OH

OH

O

OH

HO HO

E1: Hyp-1 (emodin is substrate)

Figure 3.80

Box 3.13 Hypericum/St John’s Wort The dried flowering tops of St John’s Wort (Hypericum perforatum; Guttiferae/Hypericaceae) have been used as a herbal remedy for many years, an extract in vegetable oil being employed for its antiseptic and wound healing properties. St John’s Wort is now a major crop marketed as an antidepressant, which is claimed to be as effective in its action as the widely prescribed antidepressants of the selective serotonin re-uptake inhibitor (SSRI) class such as fluoxetine (Prozac®), and with fewer side-effects. There is considerable clinical evidence that extracts of St John’s Wort are effective in treating mild to moderate depression and improving mood. However, to avoid potentially dangerous side-effects, St John’s Wort should not be used at the same time as prescription antidepressants (see below). St John’s Wort is a small to medium-height herbaceous perennial plant with numerous yellow flowers characteristic of this genus. It is widespread throughout Europe, where it is generally considered a weed, and has also become naturalized in North America. The tops, including flowers at varying stages of development which contain considerable amounts of the active principles, are harvested and dried in late summer. The dried herb contains significant amounts of phenolic derivatives, including 4–5% of flavonoids (see page 170), though the antidepressant activity is considered to derive principally from naphthodianthrone structures such as hypericin (about 0.1%) and pseudohypericin (about 0.2%), and a prenylated acylphloroglucinol derivative hyperforin (Figure 3.81). The fresh plant also contains significant levels of protohypericin and protopseudohypericin (Figure 3.81), which are converted into hypericin and pseudohypericin during drying and processing, as a result of irradiation with visible light. Hyperforin is a major

The Acetate Pathway: Fatty Acids and Polyketides 109

Box 3.13 (continued)

OH

O

OH

OH

O

OH HO

R

HO HO

OH

O

OH

O

OH

R

HO HO

R = H, hypericin R = OH, pseudohypericin

O

O

OH

O

O

hyperforin is a mixture of tautomeric forms

OH

R = H, protohypericin R = OH, protopseudohypericin

hyperforin

Figure 3.81 lipophilic constituent in the leaves and flowers (2–3%) and is now thought to be the major contributor to the antidepressant activity, as well as to the antibacterial properties of the oil extract. Studies show that the clinical effects of St John’s Wort on depression correlate well with hyperforin content. Standardized aqueous ethanolic extracts containing 0.15% hypericin and 5% hyperforin are usually employed. The aqueous solubility of hypericin and pseudohypericin is markedly increased by the presence of flavonoid derivatives in the crude extract, particularly procyanidin B2 , a dimer of epicatechin (see page 171). Hyperforin has been demonstrated to be a powerful and non-selective inhibitor of amine reuptake, thus increasing levels of serotonin, noradrenaline (norepinephrine), and dopamine; the mechanism of its inhibition is different from conventional antidepressants. Through its enolized β-dicarbonyl system, hyperforin exists as a mixture of interconverting tautomers; this reactive system leads to hyperforin being relatively unstable in solution, though salts that are stable and storable may be produced. Smaller amounts of structurally related acylphloroglucinol derivatives are also present in the herb, e.g. adhyperforin (see page 113, Figure 3.86, which also includes the biosynthesis of hyperforin). Hypericum perforatum appears to be the only Hypericum species where hyperforin is present as a major component. Hypericin also possesses extremely high toxicity towards certain viruses, a property that requires light and may arise via photo-excitation of the polycyclic quinone system. It is currently under investigation as an antiviral agent against HIV and hepatitis C. Antiviral activity appears to arise from an inhibition of various protein kinases, including those of the protein kinase C family. Hypericin and pseudohypericin are potent photosensitizers initiating photochemical reactions and are held responsible for hypericism, a photodermatosis seen in cattle that have consumed Hypericum plants present in pasture. Patients using St John’s Wort as an antidepressant need to be warned to avoid overexposure to sunlight. There is also considerable evidence that St John’s Wort interacts with a number of prescription drugs, including oral contraceptives, warfarin (anticoagulant), digoxin (cardiac glycoside), theophylline (bronchodilator), indinavir (HIV protease inhibitor), and ciclosporin (immunosuppressant). In some cases, it is known to promote the cytochrome P-450-dependent metabolism of the co-administered drugs. The action of potentially life-saving drugs may thus be seriously jeopardized by co-administration of Hypericum extracts.

lead to dermatitis and burning. Hypericin is also being investigated for its antiviral activities, in particular for its potential activity against HIV. A common feature of many natural products containing phenolic rings is the introduction of alkyl groups at nucleophilic sites. Obviously, the phenol groups themselves are nucleophilic, and with a suitable alkylating agent, O-alkyl derivatives may be formed (see page 13), e.g.

the O-methylation of emodin to physcion (Figure 3.76). However, a phenol group also activates the ring carbon atoms at the ortho and para positions, so that these positions similarly become susceptible to alkylation, leading to C-alkyl derivatives. The meta oxygenation pattern which is a characteristic feature of acetate-derived phenolics has the effect of increasing this nucleophilicity considerably, and the process of C-alkylation is very

110 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Ad H3C S

aldol reaction, aromatization

R C-methylation O

O

O

O O

O

O

SEnz

SEnz O

HO

O SAM

O

O

SEnz

CO2H OH 5-methylorsellinic acid

O

Ad H3 C S R HO

HO CO2H OH orsellinic acid

oxidation of methyl to CH2OH, then lactonization

CO2H

OPP

OH 5-methylorsellinic acid farnesyl PP

O C-alkylation

HO O OH

O

O

oxidation of side-chain double bond Ad S CH3 R HO HO2C

HO

FPP

O OH phthalide intermediate O-methylation MeO

SAM O E1

O OH demethylmycophenolic acid

O

HO2C OH mycophenolic acid

O

E1: demethylmycophenolic acid O-methyltransferase

Figure 3.82 much facilitated (see page 13). Suitable natural alkylating agents are SAM and dimethylallyl diphosphate (DMAPP). Other polyprenyl diphosphate esters may also be encountered in biological alkylation reactions (e.g. see hyperforin, page 113, vitamin K, page 182). A minor inconsistency has been discovered, in that while C-alkylation with dimethylallyl and higher diphosphates is mediated after the initial polyketide cyclization product is liberated from the enzyme, there are examples where C-methylation occurs before cyclization and is catalysed by the PKS itself (compare macrolide PKSs, page 86). 5-Methylorsellinic acid (Figure 3.82) is a simple C-methylated analogue of orsellinic acid found in Aspergillus flaviceps, and the extra methyl is derived from SAM. However, orsellinic acid (see page 101) is not a precursor of 5-methylorsellinic acid and, therefore, it is proposed that the poly-β-keto ester is methylated as part

of the reactions catalysed by the PKS (Figure 3.82). Similarly, 5-methylorsellinic acid, but not orsellinic acid, is a precursor of mycophenolic acid in Penicillium brevicompactum (Figure 3.82) [Box 3.14]. Further C-alkylation by farnesyl diphosphate (see page 210) proceeds after the aromatization step, and a phthalide intermediate is the substrate involved. The phthalide is a lactone derived from 5-methylorsellinic acid by hydroxylation of its starter methyl group and reaction with the end-of-chain carboxyl. The chain length of the farnesyl alkyl group is subsequently shortened by oxidation of a double bond, giving demethylmycophenolic acid, which is then O-methylated, again involving SAM, to produce mycophenolic acid. Note that the O-methylation step occurs only after the C-alkylations, so that the full activating benefit of two meta-positioned phenols can be utilized for the C-alkylation.

The Acetate Pathway: Fatty Acids and Polyketides 111

Box 3.14 Mycophenolic acid Mycophenolic acid (Figure 3.82) is produced by fermentation cultures of the fungus Penicillium brevicompactum. It has been known for many years to have antibacterial, antifungal, antiviral, and antitumour properties. It has recently been introduced into medicine as an immunosuppressant drug, to reduce the incidence of rejection of transplanted organs, particularly in kidney and heart transplants. It is formulated as the N-morpholinoethyl ester mycophenolate mofetil (Figure 3.83), which is metabolized after ingestion to mycophenolic acid; it is usually administered in combination with another immunosuppressant, ciclosporin (see page 454). The drug is a specific inhibitor of mammalian inosine monophosphate (IMP) dehydrogenase and has an antiproliferative activity on cells due to inhibition of guanine nucleotide biosynthesis. IMP dehydrogenase catalyses the NAD+ -dependent oxidation of IMP to xanthosine monophosphate (XMP), a key transformation in the synthesis of guanosine triphosphate (GTP) (see also caffeine biosynthesis, page 413). Rapidly growing cells have increased levels of the enzyme, so this forms an attractive target for anticancer, antiviral, and immunosuppressive therapy. Mycophenolate mofetil may also be used to treat severe refractory eczema.

O

H3CO

O N

O

O O

OH mycophenolate mofetil Figure 3.83

5 × malonyl-CoA E1 O

heterocyclic ring formation via Michael-type nucleophilic attack of OH onto enol tautomer followed by loss of leaving group

enolization

SEnz O

OH O

HO

OH OH

HO

HO

H O

OH

O O

O

OH

O

O

OH

OH

cyclization of hydroxyl onto dimethylallyl group: see furocoumarins Figures 4.33/4.34

oxidative cleavage of side-chain: see furocoumarins Figure 4.35 O

O

HO

O

O

C-alkylation with DMAPP O

HO

O E1 O

HO OH

O

OH

OH

O

visamminol

OH O

O

O

1′

O

SAM

OMe 8

1

O

3′

OMe O visnagin

OH

O

O-methylation

OPP

2

DMAPP

2′

Figure 3.84

OH O 5,7-dihydroxy2-methylchromone

hydroxylation

SAM

O

O

peucenin

4

3

5

OMe O khellin

E1: pentaketide chromone synthase

112 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition is formed from a poly-β-keto chain through Claisen condensation. The heterocyclic ring is produced by an overall dehydration reaction, formulated as a Michael-like addition reaction (see also flavonoids, page 169). Indeed, a type III PKS derived from Aloe arborescens has been characterized and shown to catalyse formation of the chromone from five molecules of malonyl-CoA; the starter unit is derived by decarboxylation. After formation of the furan ring via the C-dimethylallyl derivative peucenin and then visamminol, visnagin can be obtained by O-methylation. Alternatively, further hydroxylation para to the free phenol followed by two methylations yields khellin. The antiasthmatic properties of khellin have been exploited by developing the more polar, water-soluble derivative cromoglicate [Box 3.15].

Khellin and visnagin (Figure 3.84) are furochromones found in the fruits of Ammi visnaga (Umbelliferae/Apiaceae), and the active principles of a crude plant drug which has a long history of use as an antiasthmatic agent. Figure 3.84 presents the sequence of steps utilized in the biosynthesis of these compounds, fully consistent with the biosynthetic rationale developed above. The two carbon atoms C-2 and C-3 forming part of the furan ring originate by metabolism of a five-carbon dimethylallyl substituent attached to C-6 (for a full discussion, see furocoumarins, page 163). The 8-methoxy group in khellin is absent in visnagin, so must be introduced late in the sequence. The key intermediate is thus 5,7-dihydroxy-2-methylchromone. On inspection, this has the alternate acetate-derived oxygenation pattern and a methyl chain starter, so

Box 3.15 Khellin and Cromoglicate The dried ripe fruits of Ammi visnaga (Umbelliferae/Apiaceae) have a long history of use in the Middle East as an antispasmodic and for the treatment of angina pectoris. The drug contains small amounts of coumarin derivatives, e.g. visnadin (Figure 3.85) (compare Ammi majus, a rich source of furocoumarins, page 165), but the major constituents (2–4%) are furochromones, including khellin, visnagin, khellol, and khellol glucoside (Figure 3.85). Both khellin and visnadin are coronary vasodilators and spasmolytic agents, with visnadin actually being the more potent agent. Khellin has been used in the treatment of angina pectoris and bronchial asthma. The synthetic analogue cromoglicate (cromoglycate; Figure 3.85) is a most effective and widely used agent for the treatment and prophylaxis of asthma, hay fever, and allergic rhinitis. Cromoglicate contains two chromone systems with polar carboxylic acid functions, joined by a glycerol linker. The mode of action is not fully established. It was believed to prevent the release of bronchospasm mediators by stabilizing mast cell membranes, but an alternative suggestion is that it may act by inhibiting the effect of sensory nerve activation, thus interfering with bronchoconstriction. It is poorly absorbed orally and is thus admistered by inhalation or nasal spray. Eyedrops for relief of allergic conjunctivitis and nasal sprays for prophylaxis of allergic rhinitis are also available. The more potent pyridonochromone nedocromil (Figure 3.85) has also been introduced. OMe

O

O OAc O

O

OMe O

O

O

O

OMe O

OMe O

khellin

visnagin

O

O

R = H, khellol R = Glc, khellol glucoside O

OH visnadin

O

O

O

O

cromoglicate (cromoglycate)

Figure 3.85

OR

OMe O

O O2C

O

CO2

Et O2C

N

O

O

O

2Na

CO2 2Na

nedocromil

The Acetate Pathway: Fatty Acids and Polyketides 113 starter is formed from the amino acid valine, as seen in the pathways to bacterial iso-fatty acids (see page 56). Successive C-alkylations with DMAPP substitute both of the remaining aromatic sites. The next alkylating agent is geranyl diphosphate, and though the mechanism is exactly the same, this alkylation must destroy the aromaticity. One more alkylation occurs; this is best formulated as a carbocation addition to the geranyl double bond, generating a tertiary carbocation, then quenching

The polyketide nature of hyperforin (Figure 3.86) is almost entirely obscured by the added isoprenoid fragments. This compound is the predominant antidepressive agent in St John’s Wort (Hypericum perforatum; Guttiferae/Hypericaceae) [Box 3.13], where it co-occurs with the naphthodianthrone hypericin (see page 108). The product of the type III PKS is phlorisobutyrophenone, an analogue of phloroacetophenone (see page 100) but using isobutyryl-CoA as starter instead of acetyl-CoA. This

HO2C

DMAPP

NH2

PPO

L-Val

E1 E2

O

SEnz

3 × malonyl-CoA

HO

OH

DMAPP

O

CoAS E3

O

C-alkylation

E3

O

HO

OH

E4

OH

O

isobutyryl-CoA

O

OH

O

phlorisobutyrophenone DMAPP C-alkylation

HO

O

HO

O

HO

OH

C-alkylation

OH

C-alkylation

O

O

O

HO

O

OPP hyperforin

PPO GPP O O O H HO hyperforin

E1: E2: E3: E4:

transaminase branched-chain keto acid dehydrogenase isobutyrophenone synthase phlorisobutyrophenone dimethylallyltransferase

Figure 3.86

HO2C NH2 isoleucine

CoAS O 2-methylbutyryl-CoA

O adhyperforin

114 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O HO2C NH2

E1

O

E2

SEnz

3 × malonyl-CoA

CoAS

OH

E3

E3

O

OH

O

O

isovaleryl-CoA

L-Leu

HO

O

phlorisovalerophenone

2 × DMAPP

E1: transaminase E2: branched-chain keto acid dehydrogenase E3: phlorisovalerophenone synthase

OH O

HO

OH

HO

O2

OH

O

OH

O

humulone

deoxyhumulone

Figure 3.87

O SEnz O

O

O

C-methylation prior to cyclization

O

O

SAM

O

O

O

O

OH

O

O

SEnz

O

HO OH methylphloracetophenone

SEnz

one-electron oxidation to radical OH

OH

O

– H+, – 1e

O

OH

O

resonance forms of radical HO

HO

O

O

C

OH

keto–enol tautomerism can restore aromaticity only in the left-hand ring

O

HO

HO

O

B

A

radical coupling HO

OH

O

OH

O

O

O

H HO HO (C) O

O O

HO

OH nucleophilic attack onto enone system O

OH HO

HO

O O HO

OH O

(B)

(+)-isousnic acid

OH HO

O

OH

O

O

− H2O HO

O O

O

OH H

Figure 3.88

HO

O

HO

O O

OH O

O (+)-usnic acid (preferred tautomer)

The Acetate Pathway: Fatty Acids and Polyketides 115 this by yet another alkylation reaction on the ring system. The structurally related adhyperforin (Figure 3.86) is also found in H. perforatum. This compound is characterized by derivation from isoleucine rather than valine, and the starter unit is thus 2-methylbutyryl-CoA (compare iso-fatty acids, page 56). Somewhat less complex structures are found in hops (Humulus lululus; Cannabaceae), where they are representative of a group of compounds termed bitter acids. These are responsible for the typical bitter taste of beer, and also provide foam-stabilizing and antibacterial properties. Humulone (Figure 3.87) is typically the major bitter acid, and is formed by oxidative transformaton of deoxyhumulone. Here, the starter unit for the polyketide is leucine-derived isovaleryl-CoA. C-Methylation also features in the biosynthesis of usnic acid (Figure 3.88), an antibacterial metabolite found in many lichens, e.g. Usnea and Cladonia species, which are symbiotic combinations of alga and fungus. However, the principal structural modification encountered involves phenolic oxidative coupling (see page 28). Two molecules of methylphloracetophenone are incorporated, and these are known to derive from a pre-aromatization methylation reaction, not by methylation of phloracetophenone O

O

O

(Figure 3.88). The two molecules are joined together by an oxidative coupling mechanism which can be rationalized via the one-electron oxidation of a phenol group in methylphloracetophenone giving radical A, for which resonance forms B and C can be written. Coupling of B and C occurs. Only the left-hand ring can subsequently be restored to aromaticity by keto–enol tautomerism; this is not possible in the right-hand ring because coupling occurred on the position para to the original phenol and this position already contains a methyl. Instead, a heterocyclic ring is formed by attack of the phenol onto the enone system (see khellin, above). The outcome of this reaction is enzyme controlled; two equivalent phenol groups are present as potential nucleophiles, and two equivalent enone systems are also available. Potentially, four different products could be formed, but only one is typically produced. Loss of water then leads to usnic acid. Usnic acid is found in both (+)- and (−)-forms in nature, according to source. The product from using the alternative hydroxyl nucleophile in heterocyclic ring formation is isousnic acid, also known as a natural product in some species of lichens. Usnic acid is used as an antibacterial and preservative in toiletries.

O

OH

OH

O

OH

OMe

2 × SAM O

O

HO

O

O SEnz Claisen

O

OH

OH

OH

MeO

benzophenone intermediate

OH

OH griseophenone C

halogenation at activated site ortho to phenol group

aldol

OMe

OH

O

phenolic oxidation OMe in both rings

OH

O

OMe

O O

MeO Cl

O

O

MeO

resonance forms

O

MeO

Cl

O OMe

O-methylation

griseophenone B

OMe O OMe

stereospecific reduction of double bond

SAM MeO Cl

NADPH O

MeO Cl

dehydrogriseofulvin

Figure 3.89

OMe O OMe

O

O O

OH

Cl

stereospecific radical coupling OH

OH

O O

MeO Cl

griseofulvin

116 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Phenolic oxidative coupling is widely encountered in natural product biosynthesis, and many other examples are described in subsequent sections. A further acetate-derived metabolite formed as a result of oxidative coupling is the antifungal agent griseofulvin (Figure 3.89) synthesized by cultures of Penicillium griseofulvin [Box 3.16]. The sequence of events leading to griseofulvin has been established in detail via feeding experiments; the pathway also includes O-methylation steps and the introduction of a halogen (chlorine) atom at one of the nucleophilic sites (Figure 3.89). Initial inspection of the structure of griseofulvin shows the alternate oxygenation pattern, and also a methyl group

which identifies the start of the polyketide chain. Cyclization of the C14 poly-β-keto chain folded as shown allows both Claisen (left-hand ring) and aldol (right-hand ring) reactions to occur, giving a benzophenone intermediate. Two selective methylations lead to griseophenone C, which is the substrate for chlorination to griseophenone B; both these compounds appear as minor metabolites in P. griseofulvin cultures. One-electron oxidations on a phenolic group in each ring give a diradical; radical coupling in its resonance form generates the basic grisan skeleton with its spiro centre. Griseofulvin is then the result of methylation of the remaining phenol group and stereospecific reduction of the double bond in dehydrogriseofulvin.

Box 3.16 Griseofulvin Griseofulvin is an antifungal agent produced by cultures of P. griseofulvum and a number of other Penicillium species, including P. janczewski, P. nigrum and P. patulum. Griseofulvin is used to treat widespread or intractable dermatophyte infections, though has to some extent been superseded by newer antifungals. It is less effective when applied topically, but it is well absorbed from the gut and is selectively concentrated into the keratin layers of the skin. It may thus be used orally to control dermatophytes such as Epidermophyton, Microsporium and Trichophyton. Treatment for some conditions, e.g. infections in fingernails, may have to be continued for several months, but the drug is generally free of side-effects. Griseofulvin is also used orally or topically to control ringworm (Tinea capitis). The antifungal action appears to be through disruption of the mitotic spindle, thus inhibiting mitosis. The drug is relatively safe for use, since the effects on mitosis in sensitive fungal cells occur at concentrations that are substantially below those required to inhibit mitosis in human cells.

Starter Groups In most of the examples of aromatics so far discussed, the basic carbon skeleton has been derived from an acetate starter group (sometimes formed via PKS decarboxylation of malonate), with malonate acting as the chain extender. The molecule has then been made more elaborate, principally via alkylation reactions and the inclusion of other carbon atoms. However, the range of natural aromatic structures which are at least partly derived from acetate is increased enormously by altering the nature of the starter group from acetate to an alternative acyl-CoA system, with malonyl-CoA again providing the chain extender. For example, the starter group for hyperforin biosynthesis is a branched-chain CoA ester derived from the amino acid leucine, and this has already been discussed (see page 113). Chalcones and stilbenes are relatively simple examples of molecules in which a suitable cinnamoylCoA C6 C3 precursor from the shikimate pathway (see page 149) is used as a starter group for the PKS system. If 4-hydroxycinnamoyl-CoA (Figure 3.90)

is chain extended with three malonyl-CoA units, the poly-β-keto chain can then be folded in two ways, allowing Claisen or aldol-type cyclizations to occur as appropriate. Chalcones, e.g. naringenin-chalcone, or stilbenes, e.g. resveratrol, are the end-products formed by the type III enzymes chalcone synthase and stilbene synthase respectively. Chalcone synthase and stilbene synthase enzymes share some 75–90% amino acid sequence identity, but cyclize the same substrate in different ways. The six-membered heterocyclic ring characteristic of most flavonoids, e.g. naringenin, is formed by nucleophilic attack of a phenol group from the acetate-derived ring onto the α,β-unsaturated ketone of the chalcone (compare khellin, page 111). Stilbenes, such as resveratrol, incorporate the carbonyl carbon of the cinnamoyl unit into the aromatic ring and typically lose the end-of-chain carboxyl by a decarboxylation reaction. Although some related structures, e.g. lunularic acid (Figure 3.90) from the liverwort Lunularia cruciata, still contain this carboxyl, these are rare in nature. Carboxylated stilbenes are not intermediates in the

The Acetate Pathway: Fatty Acids and Polyketides 117 OH

OH

3 × malonyl-CoA

O

O

≡

O

CoAS

E1, E2

O 4-hydroxycinnamoyl-CoA

OH

SEnz

O

O

O

O Claisen enolization

E1 Michael-type nucleophilic addition onto α,βHO unsaturated ketone

OH

O

CO2H O

naringenin-chalcone (chalcone) CO2H

E3

OH lunularic acid

HO

E1: chalcone synthase (naringenin-chalcone synthase) E2: stilbene synthase (resveratrol synthase) E3: chalcone isomerase

E2

– CO2

OH

OH

O

OH

aldol ester hydrolysis OH

OH

O

OH

O E2

OH

OH

SEnz

HO

O

OH resveratrol (stilbene)

naringenin (flavonoid)

Figure 3.90

chalcone-type cyclization (Claisen reaction). Through appropriate amino acid replacements it is possible to convert chalcone synthase into proteins with stilbene synthase activity. Flavonoids and stilbenes, and the further modifications that may occur, are traditionally treated as shikimate-derived compounds; they are discussed in more detail in Chapter 4 (see page 167).

stilbene synthase reaction. Mutagenesis studies have confirmed that hydrolysis of the thioester bond and decarboxylation precede aromatization by the necessary dehydration/enolization reactions. Indeed, it appears that it is the TE activity component of the stilbene synthase enzyme that is primarily responsible for the stilbene-type cyclization (aldol reaction) rather than the O SCoA

malonyl-CoA

NH2

NH2

anthraniloyl-CoA O

3× SCoA malonyl-CoA

NHMe N-methyl anthraniloyl-CoA

Figure 3.91

OH

O

E1

E1: acridone synthase

N O H 4-hydroxy-2-quinolone (quinoline alkaloid)

O SEnz

O O H NMe EnzS

O

O

E1 O

N Me

OH

OH

1,3-dihydroxy-N-methylacridone (acridine alkaloid)

118 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Anthranilic acid (2-aminobenzoic acid) (see page 141) is another shikimate-derived compound which, as its CoA ester anthraniloyl-CoA, can act as a starter unit for malonate chain extension. Aromatization of the acetate-derived portion then leads to quinoline or acridine alkaloids, according to the number of acetate units incorporated (Figure 3.91). Acridone synthase is a type III PKS catalysing chain extension of N-methylanthraniloyl-CoA to give the corresponding acridine alkaloid. It is closely related structurally to chalcone synthase; indeed, it also displays a modest chalcone synthase activity. Replacement of amino acids

in three critical positions was sufficient to change its activity completely to chalcone synthase, so that it no longer accepted N-methylanthraniloyl-CoA as substrate. These products are usually considered as alkaloids, and are discussed in more detail in Chapter 6 (see page 395). Fatty acyl-CoA esters are similarly capable of participating as starter groups. Fatty acid biosynthesis and aromatic polyketide biosynthesis are distinguished by the sequential reductions as the chain length increases in the former, and by the stabilization of a reactive poly-β-keto chain in the latter, with little or no reduction involved. It is thus interesting to see natural product structures

Box 3.17 Poison Ivy and Poison Oak Poison ivy (T. radicans or Rhus radicans; Anacardiaceae) is a woody vine with three-lobed leaves that is common in North America and also found in South America. The plant may be climbing, shrubby, or may trail over the ground. It presents a considerable hazard to humans should the sap, which exudes from damaged leaves or stems, come into contact with the skin. The sap sensitizes most individuals, producing delayed contact dermatitis after a subsequent encounter. This results in watery blisters that break open, with the fluid quickly infecting other parts of the skin. The allergens may be transmitted from one person to another on the hands, on clothing, or by animals. The active principles are urushiols, a mixture of alkenyl polyphenols. In poison ivy, these are mainly catechols with C15 side-chains carrying varying degrees of unsaturation (8 , 8,11 , 8,11,14 ). Small amounts of C17 side-chain analogues are present. These catechols become oxidized to an ortho-quinone, which is then attacked by nucleophilic groups in proteins to yield an antigenic complex. Poison oak (T. toxicaria or Rhus toxicodendron; Anacardiaceae) is nearly always found as a low-growing shrub and has lobed leaflets similar to those of oak. It is also common throughout North America. There appears considerable confusion over nomenclature, and Rhus radicans may also be termed poison oak, and R. toxicodendron oakleaf poison ivy. Poison oak contains similar urushiol structures in its sap as poison ivy, though heptadecylcatechols (i.e. C17 side-chains) predominate over pentadecylcatechols (C15 side-chains). Related species of Toxicodendron, e.g. T. diversilobum (Pacific poison oak) and T. vernix (poison sumach, poison alder, poison dogwood), are also allergenic with similar active constituents. These plants were all formerly classified under the genus Rhus, but the allergen-containing species have been reclassified as Toxicodendron; this nomenclature is not widely used. Dilute purified extracts containing urushiols may be employed to stimulate antibody production and, thus, build up some immunity to the allergens. These plants cause major problems to human health in North America, with many thousands of reported cases each year.

reduction

3 × malonyl-CoA SCoA O

palmitoleoyl-CoA

O

EnzS O – CO2 hydroxylation

aldol

HO 22:1 ω7-urushiol

Figure 3.92

O

HO2C OH

22:1 ω7-anacardic acid

OH

The Acetate Pathway: Fatty Acids and Polyketides 119 oak (Toxicodendron toxicaria) [Box 3.17]. Thus, palmitoleoyl-CoA (9 -hexadecenoyl-CoA) can act as starter group for extension by three malonyl-CoA units, with a reduction step during chain extension (Figure 3.92). Aldol cyclization then gives an anacardic

containing both types of acetate/malonate-derived chains (see also zearalenone, page 89). In plants of the Anacardiaceae, just such a pathway accounts for the formation of contact allergens called urushiols in poison ivy (Toxicodendron radicans) and poison

O O

3 × malonyl-CoA

O O

CoAS

OH

HO

O

hexanoyl-CoA

CO2H

aldol

SEnz

olivetolic acid OPP

E1

geranyl PP oxidation to allylic cation; allows change in configuration around double bond (see below)

electrophilic cyclization onto carbocation

OH

OH

CO2H H

FAD E2, E3

OH

– H+ a

CO2H HO cannabigerolic acid

OH b

E2

E3

nucleophilic attack of hydroxyl onto carbocation

OH

sequential oxidation of cyclohexene gives aromatic ring

OH CO2H O

decarboxylations facilitated by ortho hydroxyl

– CO2

OH

CO2H O

tetrahydrocannabinolic acid (THC acid)

nonenzymic

OH

CO2H

OH cannabidiolic acid (CBD acid) – CO2

OH

E

CO2H

a b

C-alkylation

nonenzymic

cannabinolic acid (CBN acid) – CO2

OH

O

OH cannabidiol CBD

tetrahydrocannabinol THC

E1: olivetolate geranyltransferase

nonenzymic OH

O cannabinol CBN

E2: cannabidiolic acid (CBDA) synthase E3: tetrahydrocannabinolic acid (THCA) synthase

E/Z isomerization in geranyl substituent: E

E O

Figure 3.93

Z

120 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition acid, which is likely to be the precursor of a urushiol by decarboxylation/hydroxylation. However, enzymic evidence is not yet available. It is likely that different fatty acyl-CoAs can participate in this sequence, since urushiols from poison ivy can contain up to three double bonds in the C15 side-chain, whilst those from poison oak also have variable unsaturation in a C17 side-chain (i.e. a C18 acyl-CoA starter). The name anacardic acid relates particularly to the presence of large quantities of these compounds in the shells of cashew nuts (Anacardium occidentale; Anacardiaceae); these anacardic acids have C15 side-chains carrying one, two, or three double bonds. A saturated C6 hexanoate starter unit is used in the formation of the cannabinoids, a group of terpenophenolics found in Indian hemp (Cannabis sativa; Cannabaceae). This plant, and preparations from it, are known under a variety of names, including hashish, marihuana, pot, bhang, charas, and dagga, and have been used for centuries for the pleasurable sensations and mild euphoria experienced after its consumption, usually by smoking. The principal psychoactive component is tetrahydrocannabinol (THC; Figure 3.93), whilst structurally similar compounds such as cannabinol (CBN) and cannabidiol (CBD), present in similar or larger amounts, are effectively inactive. In recent years, the beneficial effects of cannabis, and especially THC, in alleviating nausea and vomiting in cancer patients undergoing chemotherapy, and in the treatment of glaucoma and multiple sclerosis,

has led to extensive study of cannabinoid analogues for potentially useful medicinal activity [Box 3.18]. All the cannabinoid structures contain a monoterpene C10 unit attached to a phenolic ring that carries a C5 alkyl chain. The aromatic ring/C5 chain originates from hexanoate and malonate, cyclization to a polyketide giving olivetolic acid, from which cannabigerolic acid can be obtained by C-alkylation with the monoterpene C10 unit geranyl diphosphate (Figure 3.93). Cyclization in the monoterpene unit necessitates a change in configuration of the double bond, and this involves an oxidative step in which FAD is a cofactor. It may be rationalized as involving the allylic cation, which will then allow electrophilic cyclization to proceed (compare terpenoid cyclization mechanisms, page 195). Cannabidiolic acid is then the result of proton loss, whilst tetrahydrocannabinolic acid is the product from heterocyclic ring formation via quenching with the phenol group. CBD and THC are then the respective decarboxylation products from these two compounds; decarboxylation is non-enzymic and facilitated by the adjacent phenol function (see page 22). The aromatic terpenoid-derived ring in cannabinolic acid and CBN can arise via a dehydrogenation process (compare thymol, page 204). The PKS for olivetolic acid formation has yet to be characterized; a related type III PKS system from Cannabis catalysed the formation of the decarboxylated derivative olivetol instead of olivetolic acid.

Box 3.18 Cannabis Indian hemp, Cannabis sativa (Cannabaceae), is an annual herb indigenous to central and western Asia. It is cultivated widely in India and many tropical and temperate regions for its fibre (hemp) and seed (for seed oil). The plant is also grown for its narcotic and mild intoxicant properties, and in most countries of the world, its possession and consumption are illegal. Over many years, cannabis plants have been selected for either fibre production or drug use, the former resulting in tall plants with little pharmacological activity, whilst the latter tend to be short, bushy plants. Individual plants are almost always male or female, though the sex is not distinguishable until maturity and flowering. Seeds will produce plants of both sexes in roughly equal proportions. The active principles are secreted as a resin by glandular hairs, which are more numerous in the upper parts of female plants, and resin is produced from the time flowers first appear until the seeds reach maturity. However, all parts of the plant, both male and female, contain cannabinoids. In a typical plant, the concentration of cannabinoids increases in the order: large leaves, small leaves, flowers, bracts (which surround the ovaries), with stems containing very little. Material for drug use (ganja) is obtained by collecting the flowering tops (with little leaf) from female plants, though lower quality material (bhang) consisting of leaf from both female and male plants may be employed. By rubbing the flowering tops, the resin secreted by the glandular hairs can be released and subsequently scraped off to provide cannabis resin (charas) as an amorphous brown solid or semi-solid. A potent form of cannabis, called cannabis oil, is produced by alcoholic extraction of cannabis resin. A wide variety of names are used for cannabis products according to their nature and the geographical area. In addition to the Indian words above, the names hashish (Arabia), marihuana (Europe, USA), kief and dagga (Africa) are frequently used. The term ‘assassin’ is a corruption of ‘hashishin’, a group of 13th century murderous Persians who were said to have been rewarded for their activities with hashish. Names such as grass, dope, pot, hash, weed, and wacky backy are more likely to be in current usage. Cannabis for the illicit market is cultivated worldwide. Most of the resin originates from North Africa, Afghanistan, and Pakistan, whilst

The Acetate Pathway: Fatty Acids and Polyketides 121

Box 3.18 (continued) cannabis herb comes mainly from North America and Africa. Canada, the United States, and the UK grow large quantities for legitimate use. The quantity of resin produced by the flowering tops of high-quality Indian cannabis is about 15–20%. The amount produced by various plants is dependent on several features, however, and this will markedly alter biological properties. Thus, in general, plants grown in a tropical climate produce more resin than those grown in a temperate climate. The tall fibre-producing plants are typically low resin producers, even in tropical zones. However, the most important factor is the genetic strain of the plant, as the resin produced may contain high levels of psychoactive compounds or mainly inactive constituents. The quality of any cannabis drug is potentially highly variable. The major constituents in cannabis are termed cannabinoids, a group of more than 60 structurally related terpenophenolics. The principal psychoactive agent is tetrahydrocannabinol (THC, Figure 3.93). This is variously referred to as 1 -THC or 9 -THC according to whether the numbering is based on the terpene portion or as a systematic dibenzopyran (Figure 3.94). Both systems are currently in use, though the systematic one is preferred. Also found, often in rather similar amounts, are cannabinol (CBN) and cannabidiol (CBD) (Figure 3.93), which have negligible psychoactive properties. These compounds predominate in the inactive resins. Many other cannabinoid structures have been characterized, including cannabigerol and cannabichromene (Figure 3.94). A range of cannabinoid acids, e.g. cannabidiolic acid, tetrahydrocannabinolic acid, and tetrahydrocannabinolic acid-B (Figure 3.94) are also present, as are some analogues of the other compounds mentioned, where a propyl side-chain replaces the pentyl group, e.g. tetrahydrocannabivarin (Figure 3.94). The latter compounds presumably arise from the use of butyrate rather than hexanoate as starter unit in the biosynthetic sequence. The THC content of high-quality cannabis might be in the range 0.5–1% for large leaves, 1–3% for small leaves, 3–7% for flowering tops, 5–10% for bracts, 14–25% for resin, and up to 60% in cannabis oil. Higher amounts of THC are produced in selected strains known as skunk cannabis, so named because of its powerful smell; flowering tops from skunk varieties might contain 10–15% THC. The THC content in cannabis products tends to deteriorate on storage, an effect accelerated by heat and light. Cannabis leaf and resin stored under ordinary conditions rapidly lose their activity and can be essentially inactive after about 2 years. A major change which occurs is oxidation in the cyclohexene ring resulting in conversion of THC into CBN. THC is more potent when smoked than when taken orally, its volatility allowing rapid absorption and immediate effects, so smoking has become the normal means of using cannabis. Any cannabinoid acids will almost certainly be decarboxylated upon heating, and thus the smoking process will also effectively increase somewhat the levels of active cannabinoids available, e.g. THC acid → THC (Figure 3.93). The smoking of cannabis produces a mild euphoria similar to alcohol intoxication, inducing relaxation, contentment, and a sense of well-being, with some changes in perception of sound and colour. However, this is accompanied by a reduced ability to concentrate and do complicated tasks, and a loss of short-term memory. Users claim cannabis is much preferable to alcohol or tobacco, insisting it does not cause dependence, withdrawal symptoms, or lead to the use of other drugs, 7 2

6 5

3

4

10

2′

O

6′

7

6a

10a

1 2

3′

8 9

10

8 1′

6

4′

O

HO

3

O

5′

numbered as substituted terpene

OH

OH

9

1

cannabigerol

4

5

cannabichromene CO2H

systematic numbering

OH OH O OH

O OH

O tetrahydrocannabivarin

O CO2H tetrahydrocannabinolic acid B

Figure 3.94

O nabilone

ajulemic acid

122 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.18 (continued) and they campaign vociferously for its legalization. However, psychological dependence does occur, and cannabis can lead to hallucinations, depression, anxiety, and panic, with the additonal risk of bronchitis and lung cancer when the product is smoked. Cannabis has been used medicinally, especially as a mild analgesic and tranquillizer, but more effective and reliable agents replaced it, and even controlled prescribing was discontinued. In recent times, cannabis has been shown to have valuable anti-emetic properties which help to reduce the side-effects of nausea and vomiting caused by cancer chemotherapeutic agents. This activity stems from THC, and has resulted in some use of dronabinol (synthetic THC) and the prescribing of cannabis for a small number of patients. The synthetic THC analogue nabilone (Figure 3.94) has been developed as an anti-emetic drug for reducing cytotoxic-induced vomiting. Some of the psychoactive properties of THC, e.g. euphoria, mild hallucinations, and visual disturbances, may be experienced as side-effects of nabilone treatment. Cannabis has also been shown to possess properties which may be of value in other medical conditions. There is now ample evidence that cannabis can give relief to patients suffering from chronic pain, multiple sclerosis, glaucoma, asthma, migraine, epilepsy, and other conditions. Many sufferers who cannot seem to benefit from any of the current range of drugs are obtaining relief from their symptoms by using cannabis, but are breaking the law to obtain this medication. Current thinking is that cannabis offers a number of beneficial pharmacological responses and that there should be legal prescribing of cannabinoids or derivatives. Clinical trials have already confirmed the value of cannabis and/or THC taken orally for the relief of chronic pain and the painful spasms characteristic of multiple sclerosis, and in reducing intraocular pressure in glaucoma sufferers. An oral spray preparation of natural THC + CBD (Sativex®) for multiple sclerosis patients has been approved in some countries. In general, cannabis is only able to alleviate the symptoms of these diseases; it does not provide a cure. The non-psychoactive CBD has been shown to have anti-inflammatory properties potentially useful in arthritis treatment. The synthetic cannabinoid ajulemic acid (Figure 3.94) is in clinical trials for treatment of arthritic pain and inflammation; it does not produce any psychotropic actions at therapeutic dosage. Two main cannabinoid receptors have been identified: CB1 , predominantly in the central nervous system, and CB2 , expressed mainly in the immune system. This was followed by discovery in animal brain tissue of a natural ligand for CB1 , namely anandamide (Figure 3.95), which is arachidonoylethanolamide; ananda is the Sanskrit word for bliss. Anandamide mimics several of the pharmacological properties of THC. The natural ligand of CB2 is 2-arachidonoylglycerol (Figure 3.95); this also interacts with CB1 , and since levels of 2-arachidonoylglycerol in the brain are some 800 times higher than those of anandamide, it is now thought to be the physiological ligand for both receptors, rather than anandamide. These two compounds are the main ligands, but other related natural compounds from mammalian brain also function in the same way. These include polyunsaturated fatty acid ethanolamides, namely dihomo-γ-linolenoyl- (20:3) and adrenoyl- (22:4) ethanolamides, O-arachidonoylethanolamine (virodhamine) and 2-arachidonylglyceryl ether (noladin ether). The identification of these endogenous materials may open up other ways of exploiting some of the desirable pharmacological features of cannabis.

OH

O

O N H

OH

anandamide (arachidonoylethanolamide)

O

OH

2-arachidonoylglycerol

Figure 3.95

Hexanoate is also the starter unit used in the formation of the aflatoxins, a group of highly toxic metabolites produced by Aspergillus flavus and Aspergillus parasiticus, and probably responsible for the high incidence of liver cancer in some parts of Africa. These compounds were first detected following the deaths of young turkeys fed on mould-contaminated

peanuts (Arachis hypogaea; Leguminosae/Fabaceae). Peanuts still remain one of the crops most likely to present a potential risk to human health because of contamination with aflatoxins [Box 3.19]. These and other food materials must be routinely screened to ensure levels of aflatoxins do not exceed certain set limits. The aflatoxin structures contain a bisfuran unit fused to an

The Acetate Pathway: Fatty Acids and Polyketides 123 acetyl-CoA + 2 × malonyl-CoA NADPH

O

O CoAS hexanoyl-CoA

O O

E3

O

O versiconal acetate

H

OH OH

O OH O

E8

OH

OH

H

versicolorin B

O

O

O

(ketal)

O

averufin

A

OH

O

OH oxoaverantin

OH

hydroxyl lost via dehydration during subsequent modifications

O

several steps: rotation of left-hand ring system; formation of ether linkage to give xanthone

dihydrobisfuran

H

O

O2 NADPH O NADPH

O H O

E11

OH

versicolorin A (anthraquinone)

OH

O

H O

HO CO2H

E12

O H methylation

decarboxylation

demethylsterigmatocystin

oxidative cleavage of aromatic ring with loss of one carbon; recyclization as indicated

H

SAM

E13

O H

MeO

aflatoxin G1

O

O

E10 HO

O O

H

O

O (ester)

O2 NADPH

O

O

OH

versiconal acetate

O H tetrahydrobisfuran

HO

O

O

formation of new furan ring (acetal)

O

NAD+

E6

oxoaverantin

E7

H

versiconal

E9

H O O H

aflatoxin B1 (coumarin) E1, E2: Fas1, Fas2 (FAS) E3: PksA (PKS) E4: Nor-1 (reductase) E5: AvnA (hydroxylase) E6: AdhA (alcohol dehydrogenase)

Figure 3.96

see B; includes rearrangement and Baeyer–Villiger HO oxidation ketone → ester

oxoaverantin (≡ averufin)

O

MeO

ketal formation (non-enzymic) see A OH

acetyl carbons lost by hydrolysis

HO

O

oxidation of 5′ hydroxyl to ketone

OH O OH

O

O2, NADPH

E5

5′-hydroxyaverantin

(ester)

O OH

O

5′

E4 NADPH averantin

5′ hydroxylation

O

rearrangement details not known

OH

O

OH

1′ ketone reduction

O O H

Baeyer–Villiger oxidation

O

OH

HO

O

O (ketone) OH

H

O

1′

hemiacetal formation

O

OH

O

O

O norsolorinic acid

H

OH

SEnz

O

7 × malonyl-CoA

B

aldol and Claisen reactions; aromatizations; oxidation OH

E1, E2

O2 NADPH E15

MeO

O

HO SAM

H

O MeO

O O H

O-methylsterigmatocystin E7: AvfA (oxidase) E8: EstA (esterase) E9: VBS (VERB synthase) E10: VerB (desaturase) E11: AflN (oxidase)

E12: E13: E14: E15:

E14

O

H

O

O O H

MeO

sterigmatocystin (xanthone) AflM (reductase) OmtB (methyltransferase) OmtA (methyltransferase) OrdA (oxidoreductase)

124 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition aromatic ring, e.g. aflatoxin B1 and aflatoxin G1 , and their remarkably complex biosynthetic origin begins with a poly-β-keto chain derived from a hexanoyl-CoA starter and seven malonyl-CoA extender units (Figure 3.96). This gives an anthraquinone norsolorinic acid by the now-familiar condensation reactions, but the folding of the chain is rather different from that seen with simpler anthraquinones (see page 105). The enzyme norsolorinic acid synthase utilizes just acetyl-CoA and malonyl-CoA, and is a complex of an iterative type I PKS, together with a yeast-like type I FAS that produces the hexanoate starter (compare zearalenone, page 89). The FAS is comprised of two subunit proteins and transfers the starter to the PKS without any involvement of free hexanoyl-CoA; naturally, NADPH is a necessary cofactor for the FAS component. The six-carbon side-chain of norsolorinic acid is then oxygenated and cyclized to give the ketal averufin. The remaining pathway rapidly becomes very complicated indeed and is noteworthy for several major skeletal changes and the involvement of Baeyer–Villiger oxidations (see page 27), catalysed by cytochrome P-450-dependent monooxygenases. Versiconal acetate is another known intermediate; its formation involves a rearrangement, currently far from clear, and a Baeyer–Villiger oxidation. The latter oxidation achieves principally the transfer of a two-carbon fragment (the terminal ethyl of hexanoate) to become an ester function. These two carbon atoms can then be lost by hydrolysis, leading to formation of versicolorin B, now containing

the tetrahydrobisfuran moiety (an acetal), which is oxidized in versicolorin A to a dihydrobisfuran system. Only minimal details of the following steps are shown in Figure 3.96. They result in significant structural modifications, from the anthraquinone skeleton of versicolorin A to the xanthone skeleton of sterigmatocystin, whilst the aflatoxin end-products are coumarins. Sterigmatocystin is derived from versicolorin A by oxidative cleavage of the anthraquinone system catalysed by a cytochrome P-450-dependent enzyme and involving a Baeyer–Villiger cleavage, then recyclization through phenol groups to give a xanthone skeleton. Rotation in an intermediate leads to the angular product as opposed to a linear product. One phenol group is methylated and another phenol group is lost. This is not a reductive step, but a dehydration, achievable because the initial oxidation involves the aromatic ring and temporarily destroys aromaticity. Aflatoxin B1 formation requires oxidative cleavage of an aromatic ring in sterigmatocystin, loss of one carbon, and recyclization exploiting the carbonyl functionality; interestingly, an O-methylation step is required first, though the methoxy group so formed is subsequently lost. Aflatoxin G1 was originally thought to be derived by a Baeyer–Villiger reaction on aflatoxin B1 , but it is now known to be produced via a branch pathway from O-methylsterigmatocystin; the fine detail has yet to be established.

Box 3.19 Aflatoxins Aflatoxins are potent mycotoxins produced by the fungi A. flavus and A. parasiticus. Four main naturally occurring aflatoxins, aflatoxins B1 , B2 , G1 , and G2 (Figure 3.97), are recognized, but these can be metabolized by microorganisms and animals to other aflatoxin structures, which are also toxic. Aflatoxin B1 is the most commonly encountered member of the group, and is also the most acutely toxic and carcinogenic example. Aflatoxin B2 is a dihydro derivative of aflatoxin B1 , whilst aflatoxins G1 and G2 are an analogous pair with a six-membered lactone rather than a five-membered cyclopentenone ring. These toxins are most commonly associated with peanuts (groundnuts), maize, rice, pistachio nuts, and Brazil nuts, though other crops can be affected, and although found worldwide, they are particularly prevalent in tropical and subtropical regions. Aflatoxin M1 (Figure 3.97)

O

O

O O

O

O O

R

O

H

O MeO

O H

R = H, aflatoxin B1 R = OH, aflatoxin M1

Figure 3.97

O

O O

O

H

O MeO

O H aflatoxin B2

O O

H

O MeO

O H aflatoxin G1

O MeO

O H aflatoxin G2

The Acetate Pathway: Fatty Acids and Polyketides 125

Box 3.19 (continued) is a hydroxy derivative of aflatoxin B1 and equally toxic. It may occur in cow’s milk as a result of mammalian metabolism of aflatoxin B1 originally contaminating the animal’s food. Because these compounds fluoresce strongly under UV light, they are relatively easily detected and monitored. The aflatoxins primarily affect the liver, causing enlargement, fat deposition, and necrosis, at the same time causing cells of the bile duct to proliferate, with death resulting from irreversible loss of liver function. In the case of aflatoxin B1 , this appears to be initiated by cytochrome P-450-dependent metabolism in the body to the epoxide (Figure 3.98). The epoxide intercalates with DNA, and in so doing becomes orientated towards nucleophilic attack from guanine residues. This leads to inhibition of DNA replication and of RNA synthesis, and initiates mutagenic activity. Fortunately, endogenous glutathione is normally available to minimize damage by reacting with dangerous electrophiles. A glutathione–toxin adduct is formed in the same way (Figure 3.98), and the polar functionalities make this adduct water soluble and excretable. Aflatoxins are also known to cause hepatic carcinomas, this varying with the species of animal. The above normal incidence of liver cancer in Africa and Asia has been suggested to be linked to the increased amounts of aflatoxins found in foodstuffs, and a tolerance level of 30 ppb has been recommended. Acute hepatitis may result from food containing aflatoxin B1 at levels of the order of 0.1 ppm; levels of more than 1 ppm are frequently encountered. The biosynthesis of aflatoxins proceeds through intermediates sterigmatocystin and versicolorin (see Figure 3.96). Toxins related to these structures, but differing in aromatic substituents, are also produced by various fungi. The sterigmatocystins are synthesized by species of Aspergillus and Bipolaris, and contain a reduced bifuran fused to a xanthone, whilst the versicolorins from Aspergillus versicolor contain the same type of reduced bisfuran system but fused to an anthraquinone. Like the aflatoxins, the sterigmatocystins are acutely toxic and carcinogenic. The versicolorins are less toxic, though still carcinogenic.

N O

O

N

NH

N O

O

H O

NH2

O guanine residue in DNA

O

H N

HO2C O

toxin irreversibly bound to DNA

O

NH2 N H

SH O

H

aflatoxin B1

O H

MeO

O

NH

N O

NH2

H

HO H

NH2

O

O H MeO aflatoxin B1-epoxide Cyt P-450

N

N

O

O

CO2H glutathione

O

H N

HO2C

N H

O HO

CO2H

S

H

H O H

O H

toxin irreversibly bound to glutathione

Figure 3.98

The tetracyclines are a group of broad-spectrum antibiotics produced by species of Streptomyces, and several natural and semi-synthetic members are used clinically [Box 3.20]. They contain a linear tetracyclic skeleton of polyketide origin in which the starter group is malonamyl-CoA (Figure 3.99), i.e. the coenzyme A ester of malonate semi-amide. Thus, all carbon atoms of the

tetracycline skeleton are malonate derived, and the PKS also provides the amidotransferase activity that introduces the amino function into the starter group. The main features of the pathway (Figure 3.99) were deduced from extensive studies of mutant strains of Streptomyces aureofaciens with genetic blocks causing accumulation of mutant metabolites or the production of abnormal

126 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition malonyl-CoA Gln, ATP

reduction of 9-ketone

E1

CoAS

8 × malonyl-CoA NH2

O

SEnz

9

O

O

O

HO NADPH

O NH2

E2−5

O O malonamyl-CoA

O

O

O

O

SEnz O

O

O

O

NH2

E6

O

O

O

O

O

O

E7 SEnz O OH

OH

OH

O

NH2

6

OH

5

OH

4

OH

OH

H

OH

OH

O

5

H

OH

OH

OH

NMe2

H

O

OH

H

H

OH

B

O

OH OH O

O

OH O O

O

E1: E2: E3: E4: E5: E6:

OxyD (amidotransferase) OxyA (ketosynthase) OxyB (chain length factor) OxyC (ACP) OxyP (acyltransferase) OxyJ (9-ketoreductase)

Figure 3.99

OH

O

O OH NH2

E13 OH OH O O 4-keto-6-MPT

H

H

OH NMe2 H

HO OH

O

NADPH

O

OH NH2

E17 OH

O

OH OH O

O

oxytetracycline

further modifications as in tetracycline pathway NMe2 Cl

chlorination at activated site para to phenol O H OH

OH

NH2

tetracycline

OH

H

OH

NH2

E16

Cl HO

A

OH

transamination PLP

O

OH NMe2 H

HO

OH

OH

C

OH

5a,11a-dehydro-oxytetracycline

NMe2

OH

oxidation of 1,4-quinol E12 to quinone; hydration of keto tautomer

NH2

OH O O

O

reduction of double bond of a,bunsaturated ketone; enol form is then favoured due to conjugation

5-hydroxylation NH2

O

NH2 OH

NH2

OH

OH O O

O

4-hydroxy-6-MPT

E14 OH

NMe2

O

E11

O

SAM

NADPH

D

OH

NH2

OH

6-methylpretetramide (6-MPT)

5a,11a-dehydrotetracycline

HO

OH

NH2

O

OH

O

E8

O

E10

6-hydroxylation; E15 consider the tautomeric CH−CH=CH−C=O form

O2 NADPH

11a

O

SAM

NH2

5a

O

O

OH

OH OH OH O O O anhydrotetracycline

HO

OH

O

hydroxylation at activated centre para to phenol

pretetramide

6

NH2

C-methylation para to phenol

NH2 OH

O

E8

O

E9

SEnz

SEnz O

NH2 OH OH O OH O chlortetracycline E7: OxyK (aromatase) E8: OxyN (cyclase) E9: OxyI (cyclase) E10: OxyF (methyltransferase) E11: OxyL (oxygenase) E12: OxyG (oxygenase)

O

NH2 OH

OH

OH O O

7-chloro-4-keto-6-MPT E13: E14: E15: E16: E17:

OxyQ, OxyR (aminotransferase) OxyT (N-methyltransferase) OxyS (oxygenase) OxyE (oxygenase) TchA (reductase)

O

The Acetate Pathway: Fatty Acids and Polyketides 127 tetracylines. This organism typically produces chlortetracyline, whilst the parent compound tetracycline is in fact an aberrant product synthesized in mutants blocked in the chlorination step. The use of mutants with genetic blocks also enabled the shikimate pathway (Chapter 4) to be delineated. In that case, since a primary metabolic pathway was affected, mutants tended to accumulate intermediates and could not grow unless later components of the pathway were supplied. With the tetracyclines, a secondary metabolic pathway is involved, and the relatively broad specificity of some of the enzymes concerned allows many of the later steps to proceed even if one step, e.g. the chlorination, is not achievable. This has also proved valuable for production of some of the clinical tetracycline antibiotics. More recently, the genetic details of the pathway have been clarified, with most attention being directed towards study of oxytetracycline production in Streptomyces rimosus. Nevertheless, the bulk of the pathway is effectively the same in both organisms. The PKS is an iterative type II system, i.e. the relevant enzyme activities are provided by individual separable proteins. The appropriate poly-β-keto ester is constructed almost as expected (Figure 3.99). However, the 9-keto reductive step is catalysed after chain assembly is complete,

rather than during chain extension. This is controlled by a specific 9-ketoreductase component; reduction at the position nine carbon atoms from the carboxy terminus is a remarkably consistent feature of a number of other type II PKS systems. The tetracene ring system is built up gradually, starting with the ring at the centre of the chain fold as with anthraquinones, and the first intermediate released from the enzyme is pretetramide (Figure 3.99). This is the substrate for introduction of a methyl group at C-6, giving 6-methylpretetramide (full tetracene numbering is shown in Figure 3.100). Hydroxylation in ring A followed by oxidation gives a quinone; to accommodate the quinone, this will necessitate tautomerism in ring B to the keto form. The quinone is then substrate for hydration at the A/B ring fusion. An amine group is subsequently introduced stereospecifically into ring A by a transamination reaction, followed by di-N-methylation using SAM to give anhydrotetracycline. In the last steps, C-6 and C-4 are hydroxylated, and NADPH reduction of the C-5a/11a double bond generates oxytetracycline. Omission of the C-4 hydroxylation would provide tetracycline. The pathway to chlortetracycline involves an addition chlorination step; this occurs on 4-keto-6-methylpretetramide at the nucleophilic site para to the phenol in ring D. The subsequent steps are then identical.

Box 3.20 Tetracyclines The tetracyclines (Figure 3.100) are a group of broad-spectrum, orally active antibiotics produced by cultures of Streptomyces species. Chlortetracycline isolated from Streptomyces aureofaciens was the first of the group to be discovered, closely followed by oxytetracycline from cultures of S. rimosus. Tetracycline was found as a minor antibiotic in S. aureofaciens, but may be produced in quantity by utilizing a mutant strain blocked in the chlorination step (Figure 3.99). Similarly, the early C-6 methylation step can also be blocked, and such mutants accumulate 6-demethyltetracyclines, e.g. demeclocycline (demethylchlorotetracycline). These reactions can also be inhibited in the normal strain of S. aureofaciens by supplying cultures with either aminopterin (which inhibits C-6 methylation) or mercaptothiazole (which inhibits C-7 chlorination). Oxytetracycline from S. rimosus lacks the chlorine substituent, but has an additional 5α-hydroxyl group, introduced late in the pathway. Only minor alterations can be made to the basic tetracycline structure to modify the antibiotic activity, and these are at positions 5, 6, and 7. Other functionalities in the molecule are all essential to retain activity. Semi-synthetic tetracyclines used clinically include methacycline, obtained by a dehydration reaction from oxytetracycline, and doxycycline, via reduction of the 6-methylene in methacycline. Minocycline contains a 7-dimethylamino group and is produced by a sequence involving aromatic nitration. Lymecycline is an example of an antibiotic developed by chemical modification of the primary amide function at C-2. Tetracyclines have both amino and phenolic functions, and are thus amphoteric compounds; they are more stable in acid than under alkaline conditions. They are thus suitable for oral administration and are absorbed satisfactorily. However, because of the sequence of phenol and carbonyl substituents in the structures, they act as chelators and complex with metal ions, especially calcium, aluminium, iron, and magnesium. Accordingly, they should not be administered with foods such as milk and dairy products (which have a high calcium content), aluminium- and magnesium-based antacid preparations, iron supplements, etc., otherwise erratic and unsatisfactory absorption will occur. A useful feature of doxycycline and minocycline is that their absorptions are much less affected by metal ions. Chelation of tetracyclines with calcium also precludes their use in children

128 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 3.20 (continued) Tetracyclines R4

R3

7

R2

H

6

D

R1

H

5

OH

B

OH

O

OH OH O

7

6

8 9 10

10a

11

5a

11a

5

12

A

4a

12a

NH2 O

4 3 2 1

tetracene numbering

HO

H

H

O

R3 6β

R4 7

tetracycline

H

Me

OH

H

chlortetracycline

H

Me

OH

Cl

oxytetracycline

OH

Me

OH

H

demeclocycline

H

H

OH

H

methacycline

OH

H NMe2

doxycycline

OH

Me

H

minocycline

H

H

H

NMe2 OH

OH OH O

O

lymecycline

H N

CO2H NH2

H N

natural

H

CH2

NMe2 H N

OH

R2 6α

NMe2

C

6a

R1 5

H

semisynthetic

H

NMe2

O

OH

9

N H

NH2 OH

O

OH OH O

O

tigecycline

Figure 3.100

developing their adult teeth, and in pregnant women, since the tetracyclines become deposited in growing teeth and bone. In children, this would cause unsightly and permanent staining of teeth with the chelated yellow tetracycline. Although the tetracycline antibiotics have a broad spectrum of activity spanning Gram-negative and Gram-positive bacteria, their value has decreased as bacterial resistance has developed in pathogens such as Pneumococcus, Staphylococcus, Streptococcus, and E. coli. These organisms appear to have evolved two main mechanisms of resistance: bacterial efflux and ribosome protection. In bacterial efflux, a membrane-embedded transport protein exports the tetracycline out of the cell before it can exert its effect. Ribosomal protection releases tetracyclines from the ribosome, the site of action (see below). Nevertheless, tetracyclines are the antibiotics of choice for infections caused by Chlamydia, Mycoplasma, Brucella, and Rickettsia, and are valuable in chronic bronchitis due to activity against Haemophilus influenzae. They are also used systemically to treat severe cases of acne, helping to reduce the frequency of lesions by their effect on skin flora. There is little significant difference in the antimicrobial properties of the various agents, except for minocycline, which has a broader spectrum of activity and, being active against Neisseria meningitides, is useful for prophylaxis of meningitis. The individual tetracyclines do have varying bioavailabilities, however, which may influence the choice of agent. Tetracycline and oxytetracycline are probably the most commonly prescribed agents, whilst chlortetracycline and methacycline have both been superseded. Tetracyclines are formulated for oral application or injection, as ear and eye drops, and for topical use on the skin. Doxycycline is the agent of choice for treating Lyme disease (caused by the spirochaete Borellia burgdorferi), and also finds use as a prophylactic against malaria in areas where there is widespread resistance to chloroquine and mefloquine (see page 382). A new generation of tetracyclines known as glycylcyclines has been developed to counter resistance and provide higher antibiotic activity. The first of these in general use is tigecycline (Figure 3.100), a glycylamido derivative of minocycline. It is synthesized via nitration of minocycline, itself produced from tetracycline. Tigecycline maintains antibiotic spectrum and potency, whilst overcoming resistance arising from both ribosomal protection and efflux mechanisms. Their antimicrobial activity arises by inhibition of protein synthesis. This is achieved by interfering with the binding of aminoacyl-tRNA to acceptor sites on the ribosome by disrupting the codon–anticodon interaction (see page 422). Evidence points to a single strong binding site on the smaller 30S subunit of the ribosome. Although tetracyclines can also bind to mammalian ribosomes, there appears to be preferential penetration into bacterial cells, and there are few major side-effects from using these antibiotics.

The Acetate Pathway: Fatty Acids and Polyketides 129 O O

9

O

O

O

E1

O propionyl-CoA

O

reduction of 9-ketone HO NADPH

O

9 × malonyl-CoA

CoAS

SEnz

O O

O

O

E2 O

O

O

O

O

O

O

O

E3 O

CO2H

OH

O O

O

OH

SAM

OH

OH

O

7

reduction of 7-ketone

OH

O

CO2Me

11

NADPH

OH

OH

O

OH

O

hydroxylation/oxidation O O to 13-ketone O2 NADPH OH E12

OH

OH

OH

O

OH

OH

OH

O

O

OH

HO

4

10 7

OH

HO

E1: E2: E3: E4: E5:

14

OH

13

OH

ester hydrolysis; decarboxylation − CO2

O

OH

OH

O

OH OH

O

OH

O

NH2

HO rhodomycin D

OH

O OH OH

OH

O

O

O HO

HO

NH2 doxorubicin (adriamycin)

E6: E7: E8: E9:

O

O

NH2

L-daunosamine

daunorubicin (daunomycin)

CO2Me

E11

OMe O

NH2

OH

E10

E12

DpsA, DpsB, DpsC, DpsD, DpsG DpsE (9-ketoreductase) DpsF (cyclase/aromatase) DpsY (cyclase) DnrG (oxygenase)

Figure 3.101

OH

TDP-L-daunosamine

O2 NADPH

O

O 4′

14-hydroxylation

O 13

11

O

O

HO carminomycin SAM

CO2Me

ε-rhodomycinone

OH

O NH2

E13

OH

OH

aklavinone

4

OMe O

O

E9

TDP-D-glucose

O

O

11-hydroxylation O2 NADPH

E8

aklaviketone

OH

O

esterification of carboxyl

CO2Me

O

O

aldol cyclization

O

O

O

aklanonic acid anthrone

E7

OH

O

E4

aklanonic acid E6

SEnz

O

O2 E5

O

O

CO2H

O

OH

SEnz

DnrC (O-methyltransferase) DnrD (cyclase) DnrH (reductase) DnrF (hydroxylase)

O 4′

NH2

[epimer at 4′ = epirubicin (4′-epidoxorubicin)] E10: E11: E12: E13:

DnrS (glycosyltransferase) DnrP (methylesterase) DoxA (oxygenase) DnrK (O-methyltransferase)

130 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition A number of anthracycline antibiotics, e.g. doxorubicin (Figure 3.101) from Streptomyces peuceticus and daunorubicin from Streptomyces coeruleorubicus, have structurally similar tetracyclic skeletons and would appear to be related to the tetracyclines. However, anthraquinone derivatives are intermediates in anthracycline biosynthesis, and the fourth ring is constructed later. The folding of the poly-β-keto chain is also rather different from that seen with tetracyclines; as a result, the end-of-chain carboxyl is ultimately lost through decarboxylation. This carboxyl is actually retained for a considerable portion of the pathway, and is even protected against decarboxylation by methylation to the ester, until no longer required. The starter group for the type II PKS is propionyl-CoA (Figure 3.101) and a specific 9-ketoreductase component acts upon the full-length polyketide chain, as seen with the tetracyclines. The initial enzyme-free product is the anthrone, which is oxidized to the anthraquinone alklanonic acid. The carboxylic acid is esterified, and then the fourth ring can be elaborated by a simple aldol reaction. Most of the modifications which subsequently occur

during the biosynthetic pathway are easily predictable. A feature of note in molecules such as doxorubicin and daunorubicin is the amino sugar l-daunosamine which originates from TDPglucose (thymidine diphosphoglucose; compare UDPglucose, page 31) and is introduced in the latter stages of the sequence. Hydroxylation of daunorubicin to doxorubicin is the very last step. Doxorubicin and daunorubicin are used as antitumour drugs rather than antimicrobial agents [Box 3.21]. They act primarily at the DNA level and so also have cytotoxic properties. Doxorubicin in particular is a highly successful and widely used antitumour agent, employed in the treatment of leukaemias, lymphomas and a variety of solid tumours. The production of modified aromatic polyketides by genetically engineered type II PKSs is not quite so ‘obvious’ as with the modular type I enzymes, but significant progress has been made in many systems. Each type II PKS contains a minimal set of three protein subunits, two β-ketoacyl synthase (KS) subunits and an ACP to which the growing chain is attached. Additional subunits, including KRs, cyclases (CYC), and aromatases

Box 3.21 Anthracycline Antibiotics Doxorubicin (adriamycin; Figure 3.101) is produced by cultures of S. peucetius var caesius and is one of the most successful and widely used antitumour drugs. The organism is a variant of S. peucetius, a producer of daunorubicin (see below), in which mutagen treatment resulted in expression of a latent hydroxylase enzyme and thus synthesis of doxorubicin by 14-hydroxylation of daunorubicin. Doxorubicin has one of the largest spectrum of antitumour activity shown by antitumour drugs and is used to treat acute leukaemias, lymphomas, and a variety of solid tumours. It is administered by intravenous injection and is largely excreted in the bile. The planar anthracycline molecule intercalates between base pairs on the DNA helix. The sugar unit provides further binding strength and also plays a major role in sequence recognition for the binding. Intercalation is central to doxorubicin’s primary mode of action, inhibition of the enzyme topoisomerase II, which is responsible for cleaving and resealing of double-stranded DNA during replication (see page 155). Common toxic effects include nausea and vomiting, bone marrow suppression, hair loss, and local tissue necrosis, with cardiotoxicity at higher dosage. Daunorubicin (Figure 3.101) is produced by S. coeruleorubidus and S. peucetius. Though similar to doxorubicin in its biological and chemical properties, it is no longer used therapeutically to any extent. It has a much less favourable therapeutic index than doxorubicin, and the markedly different effectiveness as an antitumour drug is not fully understood, though differences in metabolic degradation may be responsible. Epirubicin (Figure 3.101), the semi-synthetic 4 -epimer of doxorubicin, is particularly effective in the treatment of breast cancer, producing lower side-effects than doxorubicin. The antileukaemics aclarubicin from Streptomyces galilaeus, a complex glycoside of aklavinone (Figure 3.101), and the semi-synthetic idarubicin are shown in Figure 3.102. These compounds are structurally related to doxorubicin but can show increased activity with less cardiotoxicity. The principal disadvantage of all of these agents is their severe cardiotoxicity, which arises through inhibition of cardiac Na+ , K+ -ATPase. Mitoxantrone (mitozantrone) (Figure 3.102) is a synthetic analogue of the anthracyclinones in which the non-aromatic ring and the aminosugar have both been replaced with aminoalkyl side-chains. This agent has reduced toxicity compared with doxorubicin and is effective in the treatment of solid tumours and leukaemias. In addition, it is currently proving useful in multiple sclerosis treatment, where it can reduce the frequency of relapses. Despite considerable synthetic research, however, relatively few anthracycline analogues have proved superior to doxorubicin itself, though some newer ones, e.g. sabarubicin and galarubicin (Figure 3.102), are in clinical trials.

The Acetate Pathway: Fatty Acids and Polyketides 131

Box 3.21 (continued) OH

O

H N

HN

O

OH

O

OH OH O

OH

O

HN

N H

mitoxantrone (mitozantrone) OH

O O

OH

NH2 idarubicin

O

OH

OH OH

O

OH

OH

O

OH

L-daunosamine HO

NH2

O aklavinone

O

NMe2 L-rhodosamine

O OMe O

OH

L-2-deoxyfucose

O

OH O

O

OH

O

O

O

O O

OMe O

CO2Me

O HO

O

OH

OH

O

NH2 O

O HO

OH

O F

galarubicin

OH

L-2-deoxyfucose

L-cinerulose

O aclacinomycin A (aclarubicin)

sabarubicin

Figure 3.102

(ARO) are responsible for modification of the nascent chain to form the final cyclized structure. Novel polyketides have been generated by manipulating type II PKSs, exchanging KS, CYC and ARO subunits among different systems. However, because of the highly reactive nature of poly-β-keto chains, the cyclizations that occur with the modified gene product frequently vary from those in the original compound. Compared with type I PKSs, the formation of new products with predictable molecular structures has proven less controllable.

FURTHER READING Fatty Acid Synthase Jenni S, Leibundgut M, Maier T and Ban N (2006) Architecture ˚ resolution. Science 311, of a fungal fatty acid synthase at 5 A 1263–1267. Kolter T (2007) The fatty acid factory of yeasts. Angew Chem Int Ed 46, 6772–6775. Lu H and Tonge PJ (2008) Inhibitors of FabI, an enzyme drug target in the bacterial fatty acid biosynthesis pathway. Acc Chem Res 41, 11–20. Maier T, Jenni S and Ban N (2006) Architecture of mam˚ resolution. Science 311, malian fatty acid synthase at 4.5 A 1258–1262.

Smith S (2006) Architectural options for a fatty acid synthase. Science 311, 1251–1252. Smith S and Tsai S-C (2007) The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep 24, 1041–1072. Surolia A, Ramya TNC, Ramya V and Surolia N (2004) ‘FAS’t inhibition of malaria. Biochem J 383, 401–412; corrigenda 384, 655. Tasdemir D (2006) Type II fatty acid biosynthesis, a new approach in antimalarial natural product discovery. Phytochem Rev 5, 99–108. Wright HT and Reynolds KA (2007) Antibacterial targets in fatty acid biosynthesis. Curr Opin Microbiol 10, 447–453.

Fatty Acids and Fats Behrouzian B and Buist PH (2003) Bioorganic chemistry of plant lipid desaturation. Phytochem Rev 2, 103–111. Brouwer IA, Geelen A and Katan MB (2006) n–3 Fatty acids, cardiac arrhythmia and fatal coronary heart disease. Prog Lipid Res 45, 357–367. Buist PH (2004) Fatty acid desaturases: selecting the dehydrogenation channel. Nat Prod Rep 21, 249–262. Buist PH (2007) Exotic biomodification of fatty acids. Nat Prod Rep 24, 1110–1127. Chatgilialoglu C and Ferreri C (2005) Trans lipids: the free radical path. Acc Chem Res 38, 411–448. Cunnane SC (2003) Problems with essential fatty acids: time for a new paradigm? Prog Lipid Res 42, 544–568.

132 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Domergue F, Abbadi A and Heinz E (2005) Relief for fish stocks: oceanic fatty acids in transgenic oilseeds. Trends Plant Sci 10, 112–116. Drexler H, Spiekermann P, Meyer A, Domergue F, Zank T, Sperling P, Abbadi A and Heinz E (2003) Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and first results. J Plant Physiol 160, 779–802. Kaulmann U and Hertweck C (2002) Biosynthesis of polyunsaturated fatty acids by polyketide synthases. Angew Chem Int Ed 41, 1866–1869. Murphy DJ (2002) Biotechnology and the improvement of oil crops – genes, dreams and realities. Phytochem Rev 1, 67–77. Sayanova OV and Napier JA (2004) Molecules of interest. Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants. Phytochemistry 65, 147–158. Singh SP, Zhou X-R, Liu Q, Stymne S and Green AG (2005) Metabolic engineering of new fatty acids in plants. Curr Opin Plant Biol 8, 197–203. Tziomalos K, Athyros VG and Mikhailidis DP (2007) Fish oils and vascular disease prevention: an update. Curr Med Chem 14, 2622–2628. Valentine RC and Valentine DL (2004) Omega-3 fatty acids in cellular membranes: a unified concept. Prog Lipid Res 43, 383–402.

Echinacea Woelkart K and Bauer R (2008) The role of alkamides as an active principle of Echinacea. Planta Med 73, 615–623.

Lipstatin Eisenreich W, Kupfer E, Stohler P, Weber W and Bacher A (2003) Biosynthetic origin of a branched chain analogue of the lipase inhibitor, lipstatin. J Med Chem 46, 4209–4212. Schuhr CA, Eisenreich W, Goese M, Stohler P, Weber W, Kupfer E and Bacher A (2002) Biosynthetic precursors of the lipase inhibitor lipstatin. J Org Chem 67, 2257–2262.

Prostaglandins, Thromboxanes, Leukotrienes Blobaum AL and Marnett LJ (2007) Structural and functional basis of cyclooxygenase inhibition. J Med Chem 50, 1425–1441. Das S, Chandrasekhar S, Yadav JS and Gr´ee R (2007) Recent developments in the synthesis of prostaglandins and analogues. Chem Rev 107, 3286–3337. De Leval X, Hanson J, David J-L, Masereel B, Pirotte B and Dogne J-M (2004) New developments on thromboxane and prostacyclin modulators. Part II: prostacyclin modulators. Curr Med Chem 11, 1243–1252. Dogne J-M, de Leval X, Hanson J, Frederich M, Lambermont B, Ghuysen A, Casini A, Masereel B, Ruan K-H, Pirotte B and Kolh P (2004) New developments on thromboxane and prostacyclin modulators. Part I: thromboxane modulators. Curr Med Chem 11, 1223–1241. Dogn´e J-M, Supuran CT and Pratico D (2005) Adverse cardiovascular effects of the coxibs. J Med Chem 48, 2251–2257.

Fam SS and Morrow JD (2003) The isoprostanes: unique products of arachidonic acid oxidation – a review. Curr Med Chem 10, 1723–1740. Garg R, Kurup A, Mekapati SB and Hansch C (2003) Cyclooxygenase (COX) inhibitors: a comparative QSAR study. Chem Rev 103, 703–731. Jampilek J, Dolezal M, Opletalova V and Hartl J (2006) 5-Lipoxygenase, leukotrienes biosynthesis and potential antileukotrienic agents. Curr Med Chem 13, 117–129. McGinley CM and van der Donk WA (2003) Enzymatic hydrogen atom abstraction from polyunsaturated fatty acids. Chem Commun 2843–2846. Montuschi P, Barnes P and Roberts LJ (2007) Insights into oxidative stress: the isoprostanes. Curr Med Chem 14, 703–717. Murakami M and Kudo I (2004) Recent advances in molecular biology and physiology of the prostaglandin E2 -biosynthetic pathway. Prog Lipid Res 43, 3–35. Roberts SM, Santoro MG and Sickle ES (2002) The emergence of the cyclopentenone prostaglandins as important, biologically active compounds. J Chem Soc, Perkin Trans 1 1735–1742. Rouzer CA and Marnett LJ (2003) Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases. Chem Rev 103, 2239–2304. Valmsen K, J¨arving I, Boeglin WE, Varvas K, Koljak R, Pehk T, Brash AR and Samel N (2001) The origin of 15R-prostaglandins in the Caribbean coral Plexaura homomalla: molecular cloning and expression of a novel cyclooxygenase. Proc Natl Acad Sci USA 98, 7700–7705. Van der Donk WA, Tsai A-L and Kulmacz RJ (2002) The cyclooxygenase reaction mechanism. Biochemistry 41, 15451–15458. Warner TD and Mitchell JA (2002) Cyclooxygenase-3 (COX-3): filling in the gaps toward a COX continuum? Proc Natl Acad Sci USA 99, 13371–13373.

Polyketide Synthases: General Austin MB and Noel JP (2003) The chalcone synthase superfamily of type III polyketide synthases. Nat Prod Rep 20, 79–110. Cox RJ (2007) Polyketides, proteins and genes in fungi: programmed nano-machines begin to reveal their secrets. Org Biomol Chem 5, 2010–2026. Hill AM (2006) The biosynthesis, molecular genetics and enzymology of the polyketide-derived metabolites. Nat Prod Rep 23, 256–320. Liou GF and Khosla C (2003) Building-block selectivity of polyketide synthases. Curr Opin Chem Biol 7, 279–284. Moore BS and Hertweck C (2002) Biosynthesis and attachment of novel bacterial polyketide synthase starter units. Nat Prod Rep 19, 70–99. Shen B (2003) Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opin Chem Biol 7, 285–295. Staunton J and Weissman KJ (2001) Polyketide biosynthesis: a millennium review. Nat Prod Rep 18, 380–416.

The Acetate Pathway: Fatty Acids and Polyketides 133 Staunton J and Wilkinson B (2001) Combinatorial biosynthesis of polyketides and nonribosomal peptides. Curr Opin Chem Biol 5, 159–164. Walsh CT (2004) Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810. Watanabe K and Oikawa H (2007) Robust platform for de novo production of heterologous polyketides and nonribosomal peptides in Escherichia coli. Org Biomol Chem 5, 593–602.

Polyketide Synthases: Macrolides Fischbach MA and Walsh CT (2006) Assembly-line enzymology for polyketide and nonribosomal peptide antibiotics: logic, machinery, and mechanisms. Chem Rev 106, 3468–3496. Katz L and Ashley GW (2005) Translation and protein synthesis: macrolides. Chem Rev 105, 499–527. Kopp F and Marahiel MA (2007) Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Nat Prod Rep 24, 735–749. McDaniel R, Welch M and Hutchinson CR (2005) Genetic approaches to polyketide antibiotics 1. Chem Rev 105, 543– 558. Menzella HG and Reeves CD (2007) Combinatorial biosynthesis for drug development. Curr Opin Microbiol 10, 238–245. Pohl NL (2002) Nonnatural substrates for polyketide synthases and their associated modifying enzymes. Curr Opin Chem Biol 6, 773–778.

Macrolide Antibiotics Pal S (2006) A journey across the sequential development of macrolides and ketolides related to erythromycin. Tetrahedron 62, 3171–3200. Rodriguez E and McDaniel R (2001) Combinatorial biosynthesis of antimicrobials and other natural products. Curr Opin Microbiol 4, 526–534. Wu Y-J and Su W-G (2001) Recent developments on ketolides and macrolides. Curr Med Chem 8, 1727–1758.

Avermectins Ikeda H, Nonomiya T and Omura S (2001) Organization of biosynthetic gene cluster for avermectin in Streptomyces avermitilis: analysis of enzymatic domains in four polyketide synthases. J Ind Microbiol Biotechnol 27, 170–176.

Polyene Antifungals Aparicio JF, Mendes MV, Anton N, Recio E and Martin JF (2004) Polyene macrolide antibiotic biosynthesis. Curr Med Chem 11, 1645–1656. Caffrey P, Lynch S, Flood E, Finnan S and Oliynyk M (2001) Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chem Biol 8, 713–723; errata (2003) 10, 93–94. Zotchev SB (2003) Polyene macrolide antibiotics and their applications in human therapy. Curr Med Chem 10, 211– 223.

Ascomycin, Tacrolimus, Rapamycin Gregory MA, Hong H, Lill RE, Gaisser S, Petkovic H, Low L, Sheehan LS, Carletti I, Ready SJ, Ward MJ, Kaja AL, Weston AJ, Challis IR, Leadlay PF, Martin CJ, Wilkinson B and Sheridan RM (2006) Rapamycin biosynthesis: elucidation of gene product function. Org Biomol Chem 4, 3565–3568. Lowden PAS, Wilkinson B, B¨ohm GA, Handa S, Floss HG, Leadlay PF and Staunton J (2001) Origin and true nature of the starter unit for the rapamycin polyketide synthase. Angew Chem Int Ed 40, 777–779. Mann J (2001) Natural products as immunosuppressive agents. Nat Prod Rep 18, 417–430. Wu K, Chung L, Revill WP, Katz L and Reeves CD (2000) The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251, 81–90.

Epothilones Altmann K-H (2004) The merger of natural product synthesis and medicinal chemistry: on the chemistry and chemical biology of epothilones. Org Biomol Chem 2, 2137–2152. Altmann K-H and Gertsch J (2007) Anticancer drugs from nature – natural products as a unique source of new microtubule-stabilizing agents. Nat Prod Rep 24, 327–357. Feyen F, Cachoux F, Gertsch J, Wartmann M and Altmann K-H (2008) Epothilones as lead structures for the synthesis-based discovery of new chemotypes for microtubule stabilization. Acc Chem Res 41, 21–31. Nicolaou KC, Ritz´en A and Namoto K (2001) Recent developments in the chemistry, biology and medicine of the epothilones. Chem Commun 1523–1535. Rivkin A, Chou T-C and Danishefsky SJ (2005) On the remarkable antitumor properties of fludelone: how we got there. Angew Chem Int Ed 44, 2838–2850.

Rifamycins Floss HG and Yu T-W (2005) Rifamycins – mode of action, resistance, and biosynthesis. Chem Rev 105, 621–632.

Zearalenone Gaffoor I and Trail F (2006) Characterization of two polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae. Appl Environ Microbiol 72, 1793–1799. Winssinger N and Barluenga S (2007) Chemistry and biology of resorcylic acid lactones. Chem Commun 22–36.

Discodermolide Burlingame MA, Mendoza E, Ashley GW and Myles DC (2006) Synthesis of discodermolide intermediates from engineered polyketides. Tetrahedron Lett 47, 1209–1211. Smith AB and Freeze BS (2008) (+)-Discodermolide: total synthesis, construction of novel analogues, and biological evaluation. Tetrahedron 64, 261–298.

134 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Mupirocin El-Sayed AK, Hothersall J, Cooper SM, Stephens E, Simpson TJ and Thomas CM (2003) Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586. Chem Biol 10, 419–430.

Polyethers Bhatt A, Stark CBW, Harvey BM, Gallimore AR, Demydchuk Y, Spencer JB, Staunton J and Leadlay PF (2005) Accumulation of an E,E,E-triene by the monensin-producing polyketide synthase when oxidative cyclization is blocked. Angew Chem Int Ed 44, 7075–7078. Dounay AB and Forsyth CJ (2002) Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor. Curr Med Chem 9, 1939–1980. Fern´andez JJ, Candenas ML, Souto ML, Trujillo MM and Norte M (2002) Okadaic acid, useful tool for studying cellular processes. Curr Med Chem 9, 229–262. Inoue M (2004) Convergent syntheses of polycyclic ethers. Illustrations of the utility of acetal-linked intermediates. Org Biomol Chem 2, 1811–1817. Nakata T (2005) Total synthesis of marine polycyclic ethers. Chem Rev 105, 4314–4347.

Zaragozic Acids (Squalestatins) Armstrong A and Blench TJ (2002) Recent synthetic studies on the zaragozic acids (squalestatins). Tetrahedron 58, 9321–9349. Cox RJ, Glod F, Hurley D, Lazarus CM, Nicholson TP, Rudd BAM, Simpson TJ, Wilkinson B and Zhang Y (2004) Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis. Chem Commun 2260–2261.

Statins Luo J-D and Chen AF (2003) Perspectives on the cardioprotective effects of statins. Curr Med Chem 10, 1593–1601. M¨uller M (2005) Chemoenzymatic synthesis of building blocks for statin side chains. Angew Chem Int Ed 44, 362–365. Oikawa H and Tokiwano T (2004) Enzymatic catalysis of the Diels–Alder reaction in the biosynthesis of natural products. Nat Prod Rep 21, 321–352. Rajanikant GK, Zemke D, Kassab M and Majid A (2007) The therapeutic potential of statins in neurological disorders. Curr Med Chem 14, 103–112. Roche VF (2005) Antihyperlipidemic statins: a self-contained, clinically relevant medicinal chemistry lesson. Am J Pharm Educ 69, 546–560. Sorensen JL and Vederas JC (2003) Monacolin N, a compound resulting from derailment of type I iterative polyketide synthase function en route to lovastatin. Chem Commun 1492–1493.

Stocking EM and Williams RM (2003) Chemistry and biology of biosynthetic Diels–Alder reactions. Angew Chem Int Ed 42, 3078–3115.

Polyketide Synthases: Aromatics Hertweck C, Luzhetskyy A, Rebets Y and Bechthold A (2007) Type II polyketide synthases: gaining a deeper insight into enzymatic teamwork. Nat Prod Rep 24, 162–190. Rix U, Fischer C, Remsing LL and Rohr J (2002) Modification of post-PKS tailoring steps through combinatorial biosynthesis. Nat Prod Rep 19, 542–580. Thomas R (2001) A biosynthetic classification of fungal and Streptomycete fused-ring aromatic polyketides. ChemBioChem 2, 612–627. Watanabe K, Praseuth AP and Wang CCC (2007) A comprehensive and engaging overview of the type III family of polyketide synthases. Curr Opin Chem Biol 11, 279–286.

Anthraquinones Gill M (2001) The biosynthesis of pigments in Basidiomycetes. Aust J Chem 54, 721–734.

Hypericum/St John’s Wort Adam P, Arigoni D, Bacher A and Eisenreich W (2002) Biosynthesis of hyperforin in Hypericum perforatum. J Med Chem 45, 4786–4793. Barnes J, Anderson LA and Phillipson JD (2001) St John’s wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. J Pharm Pharmac 53, 583–600. Beerhues L (2006) Molecules of interest. Hyperforin. Phytochemistry 67, 2201–2207. Singh YN (2005) Potential for interaction of kava and St. John’s wort with drugs. J Ethnopharmacol 100, 108–113.

Usnic Acid Ing´olfsd´ottir K (2002) Molecules of interest. Usnic acid. Phytochemistry 61, 729–736.

Cannabis Amar MB (2006) Cannabinoids in medicine: a review of their therapeutic potential. J Ethnopharmacol 105, 1–25. Ashton JC, Wright JL, McPartland JM and Tyndall Joel DA (2008) Cannabinoid CB1 and CB2 receptor ligand specificity and the development of CB2-selective agonists. Curr Med Chem 15, 1428–1443. Drysdale AJ and Platt B (2003) Cannabinoids: mechanisms and therapeutic applications in the CNS. Curr Med Chem 10, 2719–2732. Mechoulam R, Sumariwalla PF, Feldmann M and Gallily R (2005) Cannabinoids in models of chronic inflammatory conditions. Phytochem Rev 4, 11–18.

The Acetate Pathway: Fatty Acids and Polyketides 135 Merzouki A and Mesa JM (2002) Concerning kif, a Cannabis sativa L. preparation smoked in the Rif mountains of northern Morocco. J Ethnopharmacol 81, 403–406. Raharjo TJ and Verpoorte R (2004) Methods for the analysis of cannabinoids in biological materials: a review. Phytochem Anal 15, 79–94. Taura F, Sirikantaramas S, Shoyama Y, Yoshikai K, Shoyama Y and Morimoto S (2007) Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa. FEBS Lett 581, 2929–2934.

Endocannabinoids Ahn K, McKinney MK and Cravatt BF (2008) Enzymatic pathways that regulate endocannabinoid signaling in the nervous system. Chem Rev 108, 1687–1707. Di Marzo V, Bisogno T and De Petrocellis L (2007) Endocannabinoids and related compounds: walking back and forth between plant natural products and animal physiology. Chem Biol 14, 741–756. Lambert DM and Fowler CJ (2005) The endocannabinoid system: drug targets, lead compounds, and potential therapeutic applications. J Med Chem 48, 5059–5087. Lambert DM, Vandevoorde S, Jonsson K-O and. Fowler CJ (2002) The palmitoylethanolamide family: a new class of anti-inflammatory agents? Curr Med Chem 9, 663–674. Ueda N, Okamoto Y and Tsuboi K (2005) Endocannabinoidrelated enzymes as drug targets with special reference to N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. Curr Med Chem 12, 1413–1422.

Wendeler M and Kolter T (2003) Inhibitors of endocannabinoid degradation: potential therapeutics for neurological disorders. Angew Chem Int Ed 42, 2938–2941.

Aflatoxins Bhatnagar D, Ehrlich KC and Cleveland TE (2003) Molecular genetic analysis and regulation of aflatoxin biosynthesis. Appl Microbiol Biotechnol 61, 83–93. Watanabe CMH and Townsend CA (2002) Initial characterization of a type I fatty acid synthase and polyketide synthase multienzyme complex NorS in the biosynthesis of aflatoxin B1 . Chem Biol 9, 981–988. Yabe K and Nakajima H (2004) Enzyme reactions and genes in aflatoxin biosynthesis. Appl Microbiol Biotechnol 64, 745–755.

Tetracyclines Petkovi´c H, Cullum J, Hranueli D, Hunter IS, Peri´c-Concha N, Pigac J, Thamchaipenet A, Vujaklija D and Long PF (2006) Genetics of Streptomyces rimosus, the oxytetracycline producer. Microbiol Mol Biol Rev 70, 704–728. Zhang W, Wenjun S, Wang CCC and Tang Y (2007) Investigation of early tailoring reactions in the oxytetracycline biosynthetic pathway. J Biol Chem 282, 25717–25725.

Anthracyclines Lee TS, Khosla C and Tang Y (2005) Engineered biosynthesis of aklanonic acid analogues. J Am Chem Soc 127, 12254–12262.

4

THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENYLPROPANOIDS

The shikimate pathway provides an alternative route to aromatic compounds, particularly the aromatic amino acids l-phenylalanine, l-tyrosine, and l-tryptophan. This pathway is employed by microorganisms and plants, but not by animals; accordingly, the aromatic amino acids feature among the essential amino acids for man and have to be obtained in the diet. A central intermediate in the pathway is shikimic acid (Figure 4.1), a compound which had been isolated from plants of Illicium species (Japanese ‘shikimi’) many years before its role in metabolism had been discovered. Phenylalanine and tyrosine form the basis of C6 C3 phenylpropane units found in many natural products, e.g. cinnamic acids, coumarins, lignans, and flavonoids, and along with tryptophan are precursors of a wide range of alkaloid structures. In addition, it is found that many simple benzoic acid derivatives, e.g. gallic acid and p-aminobenzoic acid (PABA; 4-aminobenzoic acid), are produced via branchpoints in the shikimate pathway. Shikimic acid is currently the raw material for synthesis of the antiviral drug oseltamivir (Tamiflu®), in demand as a defence against avian influenza (bird flu) (Figure 4.1); the main plant source is fruits of star anise (Illicium verum; Illiciaceae), though manufacturers are turning to cultures of genetically modified Escherichia coli as an alternative supply. AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS The shikimate pathway begins with a coupling of phosphoenolpyruvate (PEP) from the glycolytic pathway and d-erythrose 4-phosphate from the pentose phosphate cycle

to give the seven-carbon 3-deoxy-d-arabino-heptulosonic acid 7-phosphate (DAHP; Figure 4.2). This reaction, shown here as an aldol-type condensation, is known to be mechanistically more complex in the enzyme-catalysed version; several of the other transformations in the pathway have also been found to be surprisingly complex. Elimination of phosphoric acid from DAHP followed by an intramolecular aldol reaction generates the first carbocyclic intermediate 3-dehydroquinic acid. However, this is also an oversimplification. The elimination of phosphoric acid actually follows an NAD+ -dependent oxidation of the central hydroxyl, and this is then re-formed in an NADH-dependent reduction reaction on the intermediate carbonyl compound prior to the aldol reaction occurring. All these changes occur through the function of a single enzyme, 3-dehydroquinate synthase. Reduction of 3-dehydroquinic acid leads to quinic acid, a fairly common natural product found in the free form, as esters, or in combination with alkaloids such as quinine (see page 381). Shikimic acid itself is formed from 3-dehydroquinic acid via 3-dehydroshikimic acid through dehydration (3-dehydroquinase) and reduction (shikimate dehydrogenase) steps. According to organism, two distinct types of dehydroquinase enzymes are recognized, and these employ different reaction mechanisms. A bifunctional enzyme catalyses the two steps in plants. The simple phenolic acids protocatechuic acid (3,4-dihydroxybenzoic acid) and gallic acid (3,4,5trihydroxybenzoic acid) can be formed by branchpoint reactions from 3-dehydroshikimic acid which involve dehydration and enolization, or, in the case of gallic acid, dehydrogenation and enolization. Gallic acid features as

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

138 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

HO

O

CO2H chemically

glucose, e.g. pentagalloylglucose, whilst ellagitannins contain one or more hexahydroxydiphenic acid functions, e.g. tellimagrandin II (Figure 4.3). Hexahydroxydiphenic acid esters are formed by phenolic oxidative coupling of galloyl functions catalysed by a laccase-type phenol oxidase enzyme (Figure 4.3). Chemical hydrolysis of an ellagitannin leads to formation of the dilactone ellagic acid. The glucose ester β-glucogallin is a crucial entity in the formation of gallotannins, acting as substrate for further esterification, and also as the donor of further galloyl groups (Figure 4.3). β-Glucogallin is an activated form of gallic acid and has similar reactivity to a CoA thioester. Tannins also contribute to the astringency of foods and beverages, especially tea, coffee, and wines, through their strong interaction with salivary proteins. In addition, they have beneficial antioxidant properties (see also condensed tannins, page 171).

CO2Et

AcHN

HO OH

NH2

shikimic acid

oseltamivir Tamiflu

Figure 4.1

a component of many tannins, which are plant materials that have been used for thousands of years in the tanning of animal hides to make leather because of their ability to cross-link protein molecules. Gallotannins are esters of gallic acid with a polyalcohol, typically

OP O

HO HO PEP P O PO

DAHP OH (hemiketal form)

CO2H

aldol-type reaction E1 O

CO2H

CO2H

OH D-erythrose 4-P

OH

HO

OH DAHP CO2H

aldol-type reaction

CO2H

OH

E4

O

OH

E3

O

OH

E5

HO

OH

OH

OH

OH

shikimic acid

3-dehydroshikimic acid

3-dehydroquinic acid

quinic acid

– H2O

O

oxidation and enolization

CO2H DAHP synthase (aroF, aroG, aroH) 3-dehydroquinate synthase (aro B) 3-dehydroquinase (aro D) shikimate dehydrogenase (aroE) quinate dehydrogenase 3-dehydroshikimate dehydratase

Figure 4.2

HO CO2H NADH

OH

dehydration E6 and enolization E1: E2: E3: E4: E5: E6:

OH OH

HO CO2H – H2O

NADPH HO

O

E2

H HO

H

H

NAD+

PO O

H

HO

formally an elimination of phosphoric acid; it actually involves oxidation of the hydroxyl adjacent to the proton lost and therefore requires NAD+ cofactor; the carbonyl is subsequently reduced back to an alcohol

CO2H

HO OH protocatechuic acid

CO2H

HO

OH OH gallic acid

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 139 OH OH O

OH

OH

OH

OH OH

UDPGlc

E1

OH

HO2C

O

HO HO

O

O

E2 glucose

O β-glucogallin

gallic acid

OH O

HO HO

OH

OH

O OH

HO

O O

OH O

O

one electron oxidations of phenol groups give resonance-stabilized radicals HO O2

O 1,6-digalloylglucose β-glucogallin (3 ×)

O

OH OH

OH OH O

OH

O

O

O

O O

HO

HO

O

OH O

OH

HO OH

HO

OH HO

OH OH

pentagalloylglucose

O OH

O

hydrolysis

OH O

O O

O O

O

HO

O

OH O

radical coupling HO

OH

O

E6

O

O

OH

OH

E3−5

OH

OH

O

OH β-glucogallin

O

O O

O

OH

hydrolysis

O HO OH

glucose + gallic acid + OH HO

OH HO

OH OH spontaneous lactonization

HO2C

OH

glucose + gallic acid

OH CO2H

tellimagrandin II HO E1: gallate 1-β-glucosyltransferase E2: β-glucogallin O-galloyltransferase E3−5: galloyltransferases E6: pentagalloylglucose: oxygen oxidoreductase

OH OH

hexahydroxydiphenic acid

O

O

HO

OH

O

O

OH ellagic acid

Figure 4.3 A very important branchpoint compound in the shikimate pathway is chorismic acid (Figure 4.4), which has incorporated a further molecule of PEP as an enol ether side-chain. A simple ATP-dependent phosphorylation reaction produces shikimic acid 3-phosphate, and this is followed by an addition–elimination reaction with PEP to give 5-enolpyruvylshikimic acid 3-phosphate (EPSP). This reaction is catalysed by the enzyme EPSP synthase.

The synthetic N-(phosphonomethyl)glycine derivative glyphosate (Figure 4.4) is a powerful inhibitor of this enzyme, and is believed to bind to the PEP binding site on the enzyme. Glyphosate finds considerable use as a broad-spectrum herbicide, the plant dying because of its inability to synthesize aromatic amino acids. The transformation of EPSP to chorismic acid (Figure 4.4) involves a 1,4-elimination of phosphoric acid, though

140 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition nucleophilic attack onto protonated double bond of PEP

PEP

CO2H

CO2H

PO

ATP HO

OH

E1

HN

O P OH OH glyphosate

PO

E2

OH

PO

H

O

CO2H

– HOP 1,4-elimination of phosphoric acid

CO2H

H

OP

OH

O

1,2-elimination of phosphoric acid

CO2H

FMNH2

O P OH OH

E1: shikimate kinase (aro L) E2: EPSP synthase (aro A)

H

shikimic acid 3-P

CO2H

PEP

CO2H

CO2H

OH

OH shikimic acid CO2H

H

O

CO2H

OH chorismic acid

E3

PO

O

CO2H

OH EPSP (5-enolpyruvylshikimic acid 3-P)

E3: chorismate synthase (aro C)

Figure 4.4 this is not a simple concerted elimination. Despite the reaction involving no overall change in oxidation state, the enzyme requires reduced FMN, which is not consumed in the reaction, and this coenzyme is believed to participate via reverse transfer of an electron to the substrate. The biosynthesis of chorismic acid from phosphoenolpyruvate and d-erythrose 4-phosphate involves seven enzyme activities. In most prokaryotes, these are monofunctional activities, whilst plants utilize the bifunctional dehydroquinase–shikimate dehydrogenase activity already mentioned. Fungi, however, possess a multifunctional enzyme complex (termed AROM) that catalyses five successive transformations (reactions 2–6). 4-Hydroxybenzoic acid (Figure 4.5) is produced in bacteria from chorismic acid by an elimination reaction, losing the recently introduced enolpyruvic acid side-chain. The stereochemistry of elimination is unusually syn, and so the mechanism is perhaps more complex than at first glance. In plants, 4-hydroxybenzoic acid is formed by a branch much further on in the pathway via side-chain degradation of cinnamic acids (see page 157). The three phenolic acids so far encountered, 4-hydroxybenzoic, protocatechuic, and gallic acids, demonstrate some of the hydroxylation patterns characteristic of shikimic acid-derived metabolites, i.e. a single hydroxy para to the side-chain function, dihydroxy groups arranged ortho to each other, typically 3,4- to the side-chain, and trihydroxy groups also ortho to each other and 3,4,5- to the side-chain. The single

para-hydroxylation and the ortho-polyhydroxylation patterns contrast with the typical meta-hydroxylation patterns characteristic of phenols derived via the acetate pathway (see page 101), and in most cases allow the biosynthetic origin (acetate or shikimate) of an aromatic ring to be deduced by inspection. 2,3-Dihydroxybenzoic acid and (in microorganisms, but not in plants; see page 161) salicylic acid (2-hydroxybenzoic acid) are derived from chorismic acid via its isomer isochorismic acid (Figure 4.5). The isomerization involves an SN 2 -type of reaction, an incoming water nucleophile attacking the diene system and displacing the hydroxyl. Salicyclic acid arises by an elimination reaction analogous to that producing 4-hydroxybenzoic acid from chorismic acid, again with syn stereochemistry. In the formation of 2,3-dihydroxybenzoic acid, the side-chain of isochorismic acid is lost first by hydrolysis, then dehydrogenation of the 3-hydroxy to a 3-keto allows enolization and formation of the aromatic ring. 2,3-Dihydroxybenzoic acid is a component of the powerful iron chelator (siderophore) enterobactin (Figure 4.6) found in Escherichia coli and many other Gram-negative bacteria. Such compounds play an important role in bacterial growth by making available sufficient concentrations of essential iron. Enterobactin comprises three molecules of 2,3-dihydroxybenzoic acid and three of the amino acid l-serine, in cyclic triester form. The enzymes involved in enterobactin biosynthesis are encoded by a six-gene

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 141 elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring CO2H CO2H

E1 H

4-hydroxybenzoic acid

L-Gln E4

CO2H

O

E5

CO2H

E3

isochorismic acid

CO2H

salicylic acid

E8 oxidation of 3-hydroxyl to ketone, then enolization CO2H

NH2 O

CO2H OH OH

2,3-dihydro2,3-dihydroxybenzoic acid

2-amino-2-deoxyisochorismic acid E7 CO2H

anthranilic acid

L-Trp

NAD+

OH

E9

CO2H

NH2

NH2 p-aminobenzoic acid (PABA)

O hydrolysis of enol ether side-chain

L-Gln E6

amination using ammonia (generated from glutamine) as nucleophile

elimination of pyruvic acid

CO2H

E2

CO2H

OH chorismic acid

CO2H

NH2 4-amino-4-deoxychorismic acid PLP

OH2

O

OH

isomerization via SN2′ reaction

elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring CO2H CO2H OH OH

OH 2,3-dihydroxybenzoic acid

E1: E2: E3: E4:

chorismate lyase isochorismate synthase (EntC) salicylate synthase 4-amino-4-deoxychorismate synthase (PabA, PabB) PabA = glutamine amidotransferase E5: 4-amino-4-deoxychorismate lyase (PabC) E4 + E5: p-aminobenzoate synthase E6: 2-amino-2-deoxyisochorismate synthase E7: 2-amino-2-deoxyisochorismate lyase E6 + E7: anthranilate synthase (TrpE, TrpG) TrpG = glutamine amidotransferase E8: isochorismatase (EntB) E9: 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase (EntA)

Figure 4.5 cluster; some enzymes have more than one function. Part of this cluster encodes for the isochorismic acid to 2,3-dihydroxybenzoic acid conversion and the remainder for a modular NRPS as seen in the non-ribosomal biosynthesis of peptides (see page 438), though at a minimal level with just two modules, since only a single peptide (amide) bond is created. 2,3-Dihydroxybenzoic acid and serine are activated through an ATP-mediated mechanism prior to attachment to the enzyme complex through a thioester linkage. A phosphopantetheine linker is also used (compare fatty acid biosynthesis, page 40). The lactone-forming steps are also catalysed, with a final intramolecular lactonization to give the trimer, which is released from the enzyme. Simple amino analogues of the phenolic acids are produced from chorismic acid by related transformations in which ammonia, generated from glutamine, acts as a nucleophile (Figure 4.5). Chorismic acid can be aminated at C-4 to give 4-amino-4-deoxychorismic acid and then

p-aminobenzoic acid (PABA), or at C-2 to give the isochorismic acid analogue which will yield 2-aminobenzoic (anthranilic) acid. Amination at C-4 has been found to occur with retention of configuration, so perhaps a double inversion mechanism is involved. The syn elimination transforming 4-amino-4-deoxychorismic acid into PABA requires the cofactor pyridoxal phosphate and proceeds through an imine intermediate via the amino group. PABA forms part of the structure of folic acid (vitamin B9 ; Figure 4.7) [Box 4.1]. The folic acid structure is built up (Figure 4.7) from a dihydropterin diphosphate which reacts with PABA to give dihydropteroic acid, an enzymic step for which the sulfonamide antibiotics are inhibitors. Dihydrofolic acid is produced from the dihydropteroic acid by amide formation incorporating glutamic acid, and reduction yields tetrahydrofolic acid. This reduction step is also necessary for the continual regeneration of tetrahydrofolic acid, and forms an important site of action for some antibacterial, antimalarial, and anticancer drugs.

142 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O

O O

CO2H

O OH HO

OH 2,3-dihydroxybenzoic acid

O

CO2H O

NH2

NH

O HO

OH

L-Ser

ATP

ATP

EnzSH

EnzSH

O HN HN

O

HO

Fe3+ HO

OH

HO O

enterobactin as iron chelator

OH OH

HN O

OH

SEnz

SEnz

SEnz HO

O NH2

HO O

OH activation via AMP esters, then attachment to enzyme (see Figure 7.17) enterobactin synthase (EntBDEF) includes enzymes for isochorismate → 2,3-dihydroxybenzoate conversion (see Figure 4.5)

HO

O

O NH OH

O

HO O

N H

O O

O NH

O

O

OH OH enterobactin

OH

Figure 4.6

Box 4.1 Folic acid (Vitamin B9 ) Folic acid (vitamin B9 ; Figure 4.7) is a conjugate of a pteridine unit, p-aminobenzoic acid (PABA), and glutamic acid. It is found in yeast, liver, and green vegetables, though cooking may destroy up to 90% of the vitamin. Deficiency gives rise to anaemia, and it is also standard practice to provide supplementation during pregnancy to reduce the incidence of spina bifida. Otherwise, deficiency is not normally encountered unless there is malabsorption or chronic disease. Folic acid used for supplementation is usually synthetic. Through genetic engineering techniques it may be possible to enhance the folic acid content of common vegetables. Impressive increases (up to 25×) in folic acid content were found in fruits of tomato derived from a cross between two lines, one overexpressing the enzyme aminodeoxychorismate synthase, and the other GTP cyclohydrolase I, an enzyme from the pteridine pathway; these enzymes thus enhance the flow of metabolites through each contributing pathway. In the body, folic acid becomes sequentially reduced in the pyrazine ring portion by the enzyme dihydrofolate reductase to give dihydrofolic acid (FH2 ) and then tetrahydrofolic acid (FH4 ) (Figure 4.7). Tetrahydrofolic acid then functions as a carrier of one-carbon groups for amino acid and nucleotide metabolism. The basic ring system is able to transfer methyl, methylene, methenyl, or formyl groups by the reactions outlined in Figure 4.8 Thus, a methyl group is transferred in the regeneration of methionine from homocysteine (see page 13), purine biosynthesis involves methenyl and formyl transfer, and pyrimidine biosynthesis utilizes methylene transfer. Tetrahydrofolate derivatives also serve as acceptors of one-carbon units in degradative pathways. Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms are able to synthesize this material. This offers scope for selective action and led to the use of sulfanilamide and other antibacterial sulfa drugs, compounds that competitively inhibit dihydropteroate synthase, the biosynthetic enzyme that incorporates PABA into the structure. Specific inhibitors of dihydrofolate reductase have also become especially useful as antibacterials, e.g. trimethoprim, and antimalarial

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 143 H 2N

H2N

H2N

N

HN

H N N

ATP OH

H2N HN

E1

N

HN O

H2N OPP

N

E2 SN2 reaction

N

HN

reduction

tetrahydrofolic acid (FH4)

HN

CO2H

E3

H N

H N H N

dihydrofolic acid (FH2) E4 H 2N

N

HN

CO2H

L-Glu ATP

O

CO2H

E1: 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine diphosphokinase E2: dihydropteroate synthase E3: dihydrofolate synthase E4: dihydrofolate reductase

H N

N

N

E4

H N O

N

H2N

NADPH

H N

H N

O 7,8-dihydropteroic acid

6-hydroxymethyl7,8-dihydropterin PP

H N N H

PABA

H N

O

O 6-hydroxymethyl7,8-dihydropterin H2N

N

SO2NH2 sulfanilimide

CO2H

O

NADPH

CO2H CO2H

reduction

N N

H N H N

O

a pteridine

p-amino- O benzoic acid (PABA)

CO2H CO2H L-Glu

folic acid

Figure 4.7

Box 4.1 (continued) drugs, e.g. pyrimethamine (Figure 4.9). These agents are effective because of differences in susceptibility between the enzymes in humans and in the infective organism. Regeneration of tetrahydrofolate from dihydrofolate is vital for DNA synthesis in rapidly proliferating cells. The production of thymine from uracil requires methylation of the nucleotide deoxyuridylate (dUMP) to deoxythymidylate (dTMP) with N 5,N 10 -methylene-FH4 as the methyl donor; this is thereby transformed into dihydrofolate (Figure 4.10). To keep the reaction flowing, FH2 is reduced to FH4 , and further N 5,N 10 -methylene-FH4 is produced using a one-carbon reagent. In this process, the one-carbon reagent comes from the amino acid serine, which is transformed into glycine. Anticancer agents based on folic acid, e.g. methotrexate (Figure 4.9), also inhibit dihydrofolate reductase, but they are less selective than the antimicrobial agents and rely on a stronger binding to the enzyme than the natural substrate has. They also block pyrimidine biosynthesis. Methotrexate treatment is potentially lethal to the patient, and is usually followed by ‘rescue’ with N 5 -formyl-tetrahydrofolic acid (folinic acid, leucovorin) (Figure 4.8) to counteract the folate-antagonist action. The natural 6S isomer is termed levofolinic acid (levoleucovorin); folinic acid in drug use is usually a mixture of the 6R and 6S isomers.

144 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 4.1 (continued) H 2N

HCO2H ATP ADP

H N

N N

H2N

H N

OH

N H H

N

5

N H

OH

N

E1 HN

10

FH4

N

N OH H

L-Ser

HN O

N5-formyl-FH4 (folinic acid)

ATP E3

Gly

E2

6

N

O

N10-formyl-FH4

H N

N

H2 N

ADP E4

H 2O H N

N

H 2N

N

H2N

H N

NADP+ NADPH

N

E6

H N

N

H2N

E5 N

N

N OH

NAD+ NADH

Me HN

OH

N

N5,N10-methylene-FH4

N5-methyl-FH4

N OH

N

N

N5,N10-methenyl-FH4

E4: 5-formyltetrahydrofolate cyclo-ligase E5: methylenetetrahydrofolate reductase E6: methylenetetrahydrofolate dehydrogenase

E1: formate:tetrahydrofolate ligase E2: glycine hydroxymethyltransferase E3: methenyltetrahydrofolate cyclohydrolase

Figure 4.8 OMe N

H2N

OMe

N

N

N

N

H2N

N

H2N

N

OMe NH2

NH2 trimethoprim

N

Me N H N

NH2

Cl

pyrimethamine

O

methotrexate

CO2H CO2H

Figure 4.9

H2N

N

H N

L-Ser

Gly

H2N

N

O

H N HN

N OH

N H

N

E1 HN

N OH

FH4

E3

O

N

N

deoxyribose-P

N5,N10-methylene-FH4

dUMP E2

NADPH H2N

N

O

H N HN

E1: glycine hydroxymethyltransferase E2: thymidylate synthase E3: dihydrofolate reductase

N

N OH FH2

Figure 4.10

O HN

N deoxyribose-P dTMP

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 145 by the multi-enzyme complex tryptophan synthase. Although a precursor of l-tryptophan, anthranilic acid may also be produced by metabolism of tryptophan. Both compounds feature as building blocks for a variety of alkaloid structures (see Chapter 6). Returning to the main course of the shikimate pathway, a singular rearrangement process occurs, transforming chorismic acid into prephenic acid (Figure 4.12). This reaction, a Claisen rearrangement, transfers the PEP-derived side-chain so that it becomes directly bonded to the carbocycle and so builds up the basic C6 C3 carbon skeleton of phenylalanine and tyrosine. The reaction

Anthranilic acid (Figure 4.5) is an intermediate in the biosynthetic pathway to the indole-containing aromatic amino acid l-tryptophan (Figure 4.11). In a sequence of complex reactions, which will not be considered in detail, the indole ring system is formed by incorporating two carbon atoms from phosphoribosyl diphosphate, with loss of the original anthranilate carboxyl. The remaining ribosyl carbon atoms are then removed by a reverse aldol reaction, to be replaced on a bound form of indole by those from l-serine, which then becomes the side-chain of l-tryptophan. The last two steps shown are catalysed

CO2H NH2 anthranilic acid

CO2H

SN2 reaction

NH

CH2OP

PPO OH

O

OP

H

OH N H

reverse aldol reaction HO

O

– CO2 – H 2O

OP OH

N H

imine–enamine tautomerism

O

OH

O

enol–keto tautomerism

E3

OH OP OH

N H

E2

N H 1-(2-carboxyanilino)1-deoxyribulose 5-P

indole-3-glycerol P

CO2H HO

OP OH

CO2H CO2H

NH2 L-Ser E4

NH2

PLP

N H

OH

H

OP

OH

E4

E2

OH

H phosphoribosylanthranilic acid

HO H

N H

O

HO

phosphoribosyl PP

HO

CH2OPP

E1

OH

CO2H HO H

E1: E2: E3: E4:

N H L-Trp

indole (enzyme-bound)

anthranilate phosphoribosyltransferase (TrpD) phosphoribosylanthranilate isomerase (TrpC) indole-3-glycerol phosphate synthase (TrpC) tryptophan synthase (TrpA, TrpB)

Figure 4.11 CO2H

HO2C

CO2H

Claisen HO2C rearrangement

O

CO2H

HO2C O

O

CO2H O OH chorismic acid

Figure 4.12

CO2H

O OH

HO2C

chorismic acid (pseudoequatorial conformer)

CO2H

E1 OH chorismic acid (pseudoaxial conformer)

OH

OH

prephenic acid

prephenic acid

E1: chorismate mutase

146 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition transamination: keto acid amino acid CO2H

CO2H

O

NH2 PLP E6

decarboxylation, aromatization and loss of leaving group

phenylpyruvic acid E2

H

O

CO2H

chorismate mutase prephenate dehydratase arogenate dehydratase prephenate dehydrogenase arogenate dehydrogenase phenylpyruvate aminotransferase prephenate aminotransferase 4-hydroxyphenylpyruvate aminotransferase

CO2H

H O

O

PLP

O

NH2

E7 OH H prephenic acid

an additional oxidation step E4 (of alcohol to ketone) means OH is retained on decarboxylation and aromatization; no discrete ketone intermediate is formed E1: E2: E3: E4: E5: E6: E7: E8:

O

CO2H

E1

OH chorismic acid

decarboxylation, aromatization and loss of leaving group

E3

O CO2H

L-Phe

OH L-arogenic acid

NAD+

E5

CO2H

NAD+ CO2H

O

NH2

oxidation means OH is retained on decarboxylation and aromatization; no discrete ketone intermediate is formed

PLP E8 OH 4-hydroxyphenylpyruvic acid

OH L-Tyr

Figure 4.13 is catalysed in nature by the enzyme chorismate mutase, and although it can also occur thermally, the rate increases some 106 -fold in the presence of the enzyme. The enzyme achieves this by binding the pseudoaxial conformer of chorismic acid, allowing a transition state with chair-like geometry to develop. Pathways to the aromatic amino acids l-phenylalanine and l-tyrosine via prephenic acid may vary according to the organism, and often more than one route may operate in a particular species according to the enzyme activities which are available (Figure 4.13). In essence, only three reactions are involved, decarboxylative aromatization, transamination, and, in the case of tyrosine biosynthesis, an oxidation, but the order in which these reactions occur differentiates the routes. Decarboxylative aromatization of prephenic acid yields phenylpyruvic acid, and PLP-dependent transamination then leads to l-phenylalanine. In the presence of an

NAD+ -dependent dehydrogenase enzyme, decarboxylative aromatization occurs with retention of the hydroxyl function, though as yet there is no evidence that any intermediate carbonyl analogue of prephenic acid is involved. Transamination of the resultant 4-hydroxyphenylpyruvic acid subsequently gives l-tyrosine. l-Arogenic acid is the result of transamination of prephenic acid occurring prior to the decarboxylative aromatization, and can be transformed into both l-phenylalanine and l-tyrosine depending on the absence or presence of a suitable enzymic dehydrogenase activity. In some organisms, broad activity enzymes are known to be capable of accepting both prephenic acid and arogenic acid as substrates. In microorganisms and plants, l-phenylalanine and l-tyrosine tend to be synthesized separately, as in Figure 4.13, but in animals, which lack the shikimate pathway, direct hydroxylation of l-phenylalanine to l-tyrosine, and of l-tyrosine to l-dihydroxyphenylalanine (l-DOPA) may

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 147

O2 tetrahydroCO2H biopterin NH2

E1

CO2H

O2 tetrahydrobiopterin

NH2

HO

L-Phe

O2

CO2H

HO

O

L-DOPA nucleophilic attack onto enone

HO

CO2H

MELANINS

N H cycloDOPA

HO

DOPAchrome E1: phenylanine hydroxylase E2: tyrosine hydroxylase

(see Figure 6.39)

O2 E3 O

CO2H

CO2H

N

HO

CATECHOLAMINES noradrenaline, adrenaline

NH2

E3

L-Tyr

O

HO

E2

NH2

O

DOPAquinone

E3: tyrosinase

Figure 4.14 be achieved (Figure 4.14). These reactions are catalysed by tetrahydropterin-dependent hydroxylase enzymes, the hydroxyl oxygen being derived from molecular oxygen. l-DOPA is a precursor of the catecholamines, e.g. the neurotransmitter noradrenaline and the hormone

adrenaline (see page 335). Tyrosine and DOPA are also converted by oxidation reactions into a heterogeneous polymer melanin, the main pigment in mammalian skin, hair, and eyes. In this material, the indole system is not formed from tryptophan, but arises from DOPA

decarboxylation and aromatization; amino group is retained via an additional oxidation step (amine → imine) (see Figure 4.13) O

CO2H

CO2H

Claisen rearrangement HO2C (see Figure 4.12)

CO2H

O

O

CO2H transamination

NH2

PLP

CO2H

NH2 4-amino-4-deoxychorismic acid

NH2 4-amino-4-deoxyprephenic acid

NH2 4-aminophenylpyruvic acid

NH2 L-p-aminophenylalanine L-PAPA activation via AMP E1 ester, then attachment to enzyme E2 (see Figure 7.17)

ATP EnzSH CH2OH HO

reductive release from enzyme HO

NHCOCHCl2

NO2 chloramphenicol

Figure 4.15

O

SEnz

b-hydroxylation; N-acylation; N-oxygenation of amine to nitro

O

CHCl2COSCoA

NHCOCHCl2

NADPH

O

E3

E5

O2 2-oxoglutarate

E4

SEnz NH2

E3

NO2

NH2 E1: CmlK E2: CmlP E3: CmlA (dioxygenase)

E4: CmlG (amidase) E5: CmlI (oxygenase)

148 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition by cyclization of DOPAquinone, the nitrogen of the side-chain then attacking the ortho-quinone (Figure 4.14). In the melanin biosynthetic pathway, the enzyme tyrosinase, an O2 -dependent monophenol monooxygenase, is responsible for hydroxylation of tyrosine, oxidation of DOPA to DOPAquinone, and further steps beyond DOPAchrome. Some organisms are capable of synthesizing an unusual variant of l-phenylalanine, the aminated derivative l-p-aminophenylalanine (l-PAPA; Figure 4.15). This is known to occur by a series of reactions paralleling those in Figure 4.13, but utilizing the PABA precursor 4-amino-4-deoxychorismic acid (Figure 4.5) instead of chorismic acid. Thus, amino derivatives of prephenic acid and pyruvic acid are elaborated. Although enzymes have not been characterized, appropriate genes have been identified. One important metabolite

known to be formed from l-PAPA is the antibiotic chloramphenicol, produced by cultures of Streptomyces venezuelae [Box 4.2]. The late stages of the pathway (Figure 4.15) have been formulated by genetic analysis to involve a non-ribosomal synthase system related to NRPSs (see page 438). The enzyme contains an adenylation domain, a peptidyl carrier domain, and a domain that is homologous to an NAD+ -dependent dehydrogenase. It is proposed that activation and attachment to the enzyme is followed by β-hydroxylation and N-acylation in the side-chain, the latter reaction probably requiring a coenzyme A ester of dichloroacetic acid. This is followed by oxidation of the 4-amino group to a nitro, a fairly rare substituent in natural product structures; the final step is reductive release of the product from the enzyme.

Box 4.2 Chloramphenicol Chloramphenicol (chloromycetin; Figure 4.15) was initially isolated from cultures of Streptomyces venezuelae, but is now obtained for drug use by chemical synthesis. It was one of the first broad-spectrum antibiotics to be developed, and exerts its antibacterial action by inhibiting protein biosynthesis. It binds reversibly to the 50S subunit of the bacterial ribosome, and in so doing it disrupts peptidyl transferase, the enzyme that catalyses peptide bond formation (see page 422). This reversible binding means that bacterial cells not destroyed may resume protein biosynthesis when no longer exposed to the antibiotic. Some microorganisms have developed resistance to chloramphenicol by an inactivation process involving enzymic acetylation of the primary alcohol group in the antibiotic. The acetate binds only very weakly to the ribosomes, so has little antibiotic activity. The value of chloramphenicol as an antibacterial agent has been severely limited by some serious side-effects. It can cause blood disorders, including irreversible aplastic anaemia in certain individuals, and these can lead to leukaemia and perhaps prove fatal. Nevertheless, it is still the drug of choice for some life-threatening infections, such as typhoid fever and bacterial meningitis. The blood constitution must be monitored regularly during treatment to detect any abnormalities or adverse changes. The drug is orally active, but may also be injected. Eye-drops are useful for the treatment of bacterial conjunctivitis.

PHENYLPROPANOIDS Cinnamic Acids and Esters l-Phenylalanine and l-tyrosine, as C6 C3 building blocks, are precursors for a wide range of natural products. In plants, a frequent first step is the elimination of ammonia from the side-chain to generate the appropriate trans (E) cinnamic acid. In the case of phenylalanine, this would give cinnamic acid, whilst tyrosine could yield 4-coumaric acid (p-coumaric acid; Figure 4.16). All plants appear to have the ability to deaminate phenylalanine via the enzyme phenylalanine ammonia lyase (PAL), but the corresponding transformation of tyrosine is more restricted, being mainly limited to

members of the grass family (the Graminae/Poaceae). Whether a separate enzyme tyrosine ammonia lyase (TAL) exists in plants, or whether grasses merely have a broad specificity PAL also capable of deaminating tyrosine, is still debated. Tyrosine-specific TAL has been found in a number of bacteria. Those species that do not transform tyrosine to 4-coumaric acid synthesize this compound by direct hydroxylation of cinnamic acid, in a cytochrome P-450-dependent reaction, and tyrosine is often channelled instead into other secondary metabolites, e.g. alkaloids. Other cinnamic acids are obtained by further hydroxylation and methylation reactions, sequentially building up substitution patterns typical of shikimate pathway metabolites, i.e. an ortho oxygenation pattern

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 149 E2 elimination of ammonia CO2H

CO2H NH2

E1 L-Phe

cinnamic acid O2 NADPH

CO2H

sequence of hydroxylation and methylation reactions

E3 hydroxylation CO2H

NH2

O2 NADPH

SAM

O2 NADPH

E4

E5

E6

E2 OH

MeO

HO

OH 4-coumaric acid (p-coumaric acid)

L-Tyr

CO2H

CO2H

CO2H

CH2OH

SAM

MeO

OH ferulic acid

OH caffeic acid

OH

E5 MeO

OMe

OH

OH

5-hydroxyferulic acid

sinapic acid

CH2OH

CH2OH

MeO OH 4-hydroxycinnamyl alcohol (p-coumaryl alcohol) E1: E2: E3: E4: E5:

CO2H

phenylalanine ammonia lyase (PAL) tyrosine ammonia lyase (TAL) cinnamate 4-hydroxylase p-coumarate 3-hydroxylase caffeic acid O-methyltransferase

MeO

OMe

OH

OH

coniferyl alcohol

sinapyl alcohol

POLYMERS ×2

×n

LIGNANS

LIGNIN

Figure 4.16

CO2H

COSCoA

HSCoA R1

R2

E1

NADPH R1

OH E1: 4-coumarate:CoA ligase E2: cinnamoyl-CoA reductase

Figure 4.17

CHO

R2 OH

E2

CH2OH

NADPH R1

R2 OH

E3: cinnamyl alcohol dehydrogenase

E3

R1

R2 OH

150 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition ester facilitates the first reduction step by introducing a better leaving group (CoAS− ) for the NADPH-dependent reaction. The second reduction step, aldehyde to alcohol, utilizes a further molecule of NADPH and is reversible. The enzymes involved generally exhibit rather broad substrate specificity and transform substrates with the different substitution patterns. Some of the more common natural cinnamic acids are 4-coumaric, caffeic, ferulic, and sinapic acids

(see page 140). Methylation is catalysed by a broadspecificity methyltransferase enzyme. Substitution patterns are not necessarily elaborated completely at the cinnamic acid stage, and coenzyme A esters and aldehydes may also be substrates for aromatic hydroxylation and methylation. Reduction of cinnamic acids via coenzyme A esters and aldehydes (Figure 4.17) leads to the corresponding alcohols, precursors of lignans and lignin (see page 151). Formation of the coenzyme A HO CO2H HO

HO

COSCoA

HO

HO HO

OH

O

HO

E1

O

OH

caffeoyl-CoA

CO2H OH

OH

chlorogenic acid (5-O-caffeoylquinic acid)

quinic acid E1: quinate O-hydroxycinnamoyltransferase

COSCoA

OH

OH

OH

O

L-Tyr E3

HO2C

HO 4-coumaroyl-CoA

E2

HO2C

4-hydroxyphenyllactic acid

4-hydroxyphenylpyruvic acid

E4 O

OH

CO2H 3′

O

3

E5

HO

O

O2 NADPH

HO

E6

O

OH OH

HO rosmarinic acid

4-coumaroyl-4-hydroxyphenyllactic acid E2: tyrosine aminotransferase E3: hydroxyphenylpyruvate reductase

CO2H

E4: hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase (rosmarinic acid synthase) E5, E6: hydroxycinnamoyl-hydroxyphenyllactate 3- and 3′-hydroxylases

sinapic acid E7

OH HO HO

UDPGlc

OMe OH

O O OH O 1-O-sinapoylglucose

Me3N choline

OH Me3N

OMe E8

O

OMe O

glucose

E7: sinapate 1-glucosyltransferase E8: sinapoylglucose:choline O-sinapoyltransferase (sinapine synthase)

Figure 4.18

OMe

OH

sinapine

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 151 (Figure 4.16). These can be found in plants in free form and in a range of esterified forms, e.g. with quinic acid as in chlorogenic acid (5-O-caffeoylquinic acid; see coffee, page 414), with 3,4-dihydroxyphenyl-lactic acid as in rosmarinic acid, with glucose as in 1-O-sinapoylglucose, and with choline as in sinapine (Figure 4.18). Esterification reactions require the cinnamic acid to be transformed initially to a suitably activated form, either a coenzyme A ester or a glucose ester. Coenzyme A esters are involved in the formation of chlorogenic acid in sweet potato (Ipomoea batatas; Convolvulaceae) and rosmarinic acid in Coleus blumei (Labiatae/Lamiaceae). Glucose esters are used in the biosynthesis of sinapine in rapeseed (Brassica napus; Cruciferae/Brassicaceae) (compare gallotannins, page 139). In the case of rosmarinic acid, further hydroxylation in the aromatic rings has been found to occur after the esterification reaction. This pathway also incorporates phenylalanine into one C6 C3 unit, the ‘acid’ part of the ester, and tyrosine into the other. Rosmarinic acid, first isolated from rosemary (Rosmarinus officinalis; Labiatae/Lamiaceae), is found in a number of medicinal plants and has beneficial antioxidant, antibacterial, and anti-inflammatory properties.

Lignans and Lignin The cinnamic acids also feature in the pathways to other metabolites based on C6 C3 building blocks. Pre-eminent amongst these, certainly as far as nature is concerned, is the plant polymer lignin, a strengthening material for the plant cell wall which acts as a matrix for cellulose microfibrils (see page 494). Lignin represents a vast reservoir of aromatic materials, mainly untapped because of the difficulties associated with release of these metabolites. The action of wood-rotting fungi offers the most effective way of making these useful products more accessible. Lignin is formed by phenolic oxidative coupling of hydroxycinnamyl alcohol monomers, brought about by peroxidase enzymes (see page 28). The most important of these monomers are 4-hydroxycinnamyl alcohol (p-coumaryl alcohol), coniferyl alcohol, and sinapyl alcohol (Figure 4.16), though the monomers used vary according to the plant type. Gymnosperms polymerize mainly coniferyl alcohol, dicotyledonous plants coniferyl alcohol and sinapyl alcohol, whilst monocotyledons use all three alcohols. The peroxidase enzyme then achieves one-electron oxidation of the phenol group. One-electron oxidation of a simple phenol allows delocalization of the unpaired electron, giving resonance forms in which the free electron resides at positions ortho and para to the oxygen function (see page 28). With cinnamic acid derivatives, conjugation allows the unpaired electron to

be delocalized also into the side-chain (Figure 4.19). Radical pairing of resonance structures can then provide a range of dimeric systems containing reactive quinonemethides which are susceptible to nucleophilic attack from hydroxyl groups in the same system, or by external water molecules. Thus, coniferyl alcohol monomers can couple, generating linkages as exemplified by guaiacylglycerol β-coniferyl ether(β-arylether linkage), dehydrodiconiferyl alcohol (phenylcoumaran linkage), and pinoresinol (resinol linkage). These dimers can react further by similar mechanisms to produce a lignin polymer containing a heterogeneous series of intermolecular bondings, as seen in the various dimers. In contrast to most other natural polymeric materials, lignin appears to be devoid of ordered repeating units, though some 50–70% of the linkages are of the β-arylether type. The dimeric materials are also found in nature and are called lignans. Some authorities like to restrict the term lignan specifically to molecules in which the two phenylpropane units are coupled at the central carbon of the side-chain, e.g. pinoresinol, whilst compounds containing other types of coupling, e.g. as in guaiacylglycerol β-coniferyl ether and dehydrodiconiferyl alcohol, are then referred to as neolignans. Lignan/neolignan formation and lignin biosynthesis are catalysed by different enzymes, and a consequence of this is that natural lignans/neolignans are normally enantiomerically pure because they arise from stereochemically controlled coupling. The control mechanisms for lignin biosynthesis are less well defined, but the enzymes appear to generate products lacking optical activity. Further cyclization and other modifications can create a wide range of lignans of very different structural types. One of the most important of the natural lignans having useful biological activity is the aryltetralin lactone podophyllotoxin (Figure 4.20), which is derived from coniferyl alcohol via the dibenzylbutyrolactones matairesinol and yatein, cyclization probably occurring as shown in Figure 4.20. Matairesinol is known to arise by reductive opening of the furan rings of pinoresinol, followed by successive oxidations to yield first a lactol then a lactone. The substitution pattern in the two aromatic rings is built up further during the pathway, i.e. matairesinol → yatein, and does not arise by initial coupling of two different cinnamyl alcohol residues. The trisubstituted ring is elaborated before the methylenedioxy ring system. The latter grouping is found in many shikimate-derived natural products, and is formed by an oxidative reaction on an ortho-hydroxymethoxy pattern (see page 27). Podophyllotoxin and related lignans are found in the roots of Podophyllum species (Berberidaceae), and have clinically useful cytotoxic and anticancer activity [Box 4.3]. The

152 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH

OH

OH

OH

OH

one-electron oxidation – H+ –e

MeO OH coniferyl alcohol

MeO

MeO

MeO

MeO

O

O

O

O

A

B

C

D

resonance forms of radical

A + D

B

+ D

radical pairing

D

+ D

radical pairing

radical pairing

OH

OH

O

H OMe

HO HO

H

OH OMe

OMe O

O

HO

enolization H2O

O O OMe

OH OMe

H

H

nucleophilic attack of water onto quinonemethide

nucleophilic attack of hydroxyls onto quinonemethides

MeO O

HO

OH OMe

OH

O

OH H

HO

OMe O

OMe MeO

O

O

OH

OMe nucleophilic attack of hydroxyl onto quinonemethide

HO pinoresinol (resinol linkage)

HO HO OMe OH OMe guaiacylglycerol β-coniferyl ether (β-arylether linkage)

O HO OMe

dehydrodiconiferyl alcohol (phenylcoumaran linkage)

Figure 4.19 so-called ‘mammalian’ lignans enterolactone and enterodiol (Figure 4.21) were first discovered in human urine and were unusual in that each aromatic ring possessed only a meta hydroxyl group. They were subsequently shown to be derived from dietary plant lignans,

especially matairesinol and secoisolariciresinol, by the action of intestinal microflora. Enterolactone and enterodiol have oestrogenic activity and have been implicated as contributing to lower levels of breast cancer amongst vegetarians (see phyto-oestrogens, page 177).

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 153

H

H

H

H MeO

MeO

OH

O

H

H

HO

HO OH

these two steps probably involve ring opening to the quinonemethide followed by reduction

OH

phenolic oxidative coupling

O

O

OMe

OMe H

NADPH

H

MeO

E1 MeO

HO

NAD+ OH

OMe

OH

H

≡

E3

H

MeO HO HO (–)-secoisolariciresinol

OH (–)-secoisolariciresinol

NAD+

MeO

O

O

O

O

O

O

HO

O

O

O modification of aromatic substitution patterns

MeO OH matairesinol

MeO

O

OMe

MeO

O O O O

O2 NADPH E4

OMe

OMe podophyllotoxin

OMe

E1: pinoresinol synthase E2: pinoresinol-lariciresinol reductase E3: secoisolariciresinol dehydrogenase

OH

hydroxylation O O O O

MeO

nucleophilic attack onto quinonemethide system

OMe

OMe yatein

hydroxylation

OH

Figure 4.20

NADPH

OH

OH

MeO

E3

(+)-lariciresinol

OH

OH

MeO

HO

E2

HO

MeO oxidation to lactone

MeO

(+)-pinoresinol

O HO

H

HO

oxidation to CHO, then hemiacetal (lactol) formation MeO

MeO

H

E2

O

OH coniferyl alcohol

OH

O2 NADPH E5

OMe

OMe deoxypodophyllotoxin E4: deoxypodophyllotoxin 7-hydroxylase E5: deoxypodophyllotoxin 6-hydroxylase

O O O O

MeO

OMe OMe β-peltatin

154 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition MeO

HO

OH OH

HO

intestinal bacteria

MeO

HO

OH

O

O

OH

HO O

O

MeO

HO

OH secoisolariciresinol

MeO

HO enterodiol

OH

enterolactone

matairesinol

Figure 4.21

Box 4.3 Podophyllum Podophyllum consists of the dried rhizome and roots of Podophyllum hexandrum (Podophyllum emodi) or Podophyllum peltatum (Berberidaceae). Podophyllum hexandrum is found in India, China, and the Himalayas, and yields Indian podophyllum, whilst Podophyllum peltatum (May apple or American mandrake) comes from North America and is the source of American podophyllum. Plants are collected from the wild. Both plants are large-leafed perennial herbs with edible fruits, though other parts of the plant are toxic. The roots contain cytotoxic lignans and their glucosides, Podophyllum hexandrum containing about 5% and Podophyllum peltatum about 1%. A concentrated form of the active principles is obtained by pouring an ethanolic extract of the root into water and drying the precipitated podophyllum resin or ‘podophyllin’. Indian podophyllum yields about 6–12% of resin containing 50–60% lignans, and American podophyllum 2–8% of resin containing 14–18% lignans. OH

OH

O

O

O

O O

O

O

4′

MeO

O

OMe

MeO

OR

OR R = Me, podophyllotoxin R = H, 4′-demethylpodophyllotoxin

R = Me, β-peltatin R = H, α-peltatin O

MeO

O

O

S

O

O

O HO

O OH

O

O

O O

O O

OMe

4′-demethylepipodophyllotoxin

O O

O

OH

OMe

OMe podophyllotoxone

OH

O

MeO

OMe

deoxypodophyllotoxin

HO

OH

O

OMe

O

Figure 4.22

O

O

OMe

O

O

O

MeO

O

O

O

O

MeO

4′

OR R = H, etoposide R = P, etopophos

OMe

O

MeO

OMe OH

teniposide

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 155

Box 4.3 (continued) The lignan constituents of the two roots are the same, but the proportions are markedly different. The Indian root contains chiefly podophyllotoxin (Figure 4.22) (about 4%) and 4 -demethylpodophyllotoxin (about 0.45%). The main components in the American root are podophyllotoxin (about 0.25%), β-peltatin (about 0.33%) and α-peltatin (about 0.25%). Deoxypodophyllotoxin and podophyllotoxone are also present in both plants, as are the glucosides of podophyllotoxin, 4 -demethylpodophyllotoxin, and the peltatins, though preparation of the resin results in considerable losses of the water-soluble glucosides. Podophyllum resin has long been used as a purgative, but the discovery of the cytotoxic properties of podophyllotoxin and related compounds has now made podophyllum a commercially important drug plant. Preparations of podophyllum resin (the Indian resin is preferred) are effective treatments for warts, and pure podophyllotoxin is available as a paint for venereal warts, a condition which can be sexually transmitted. The antimitotic effect of podophyllotoxin and the other lignans is by binding to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules (compare vincristine, page 375, and colchicine, page 361). During mitosis, the chromosomes separate with the assistance of these microtubules, and after cell division, the microtubules are transformed back to tubulin. Podophyllotoxin and other Podophyllum lignans were found to be unsuitable for clinical use as anticancer agents due to toxic side-effects, but the semi-synthetic derivatives etoposide and teniposide (Figure 4.22), which are manufactured from natural podophyllotoxin, have proved excellent antitumour agents. They were developed as modified forms (acetals) of the natural 4 -demethylpodophyllotoxin glucoside. Attempted synthesis of the glucoside inverted the stereochemistry at the sugar–aglycone linkage, and these agents are thus derivatives of 4 -demethylepipodophyllotoxin (Figure 4.22). Etoposide is a very effective anticancer agent, and is used in the treatment of small-cell lung cancer, testicular cancer, and lymphomas, usually in combination therapies with other anticancer drugs. It may be given orally or intravenously. The water-soluble pro-drug etopophos (etoposide 4 -phosphate) is also available; this is efficiently converted into etoposide by phosphatase enzymes and is preferred for routine clinical use. Teniposide has similar anticancer properties and, though not as widely used as etoposide, has value in paediatric neuroblastoma. Remarkably, the 4 -demethylepipodophyllotoxin series of lignans do not act via a tubulin-binding mechanism as does podophyllotoxin. Instead, these drugs inhibit the enzyme topoisomerase II, thus preventing DNA synthesis and replication. Topoisomerases are responsible for cleavage and resealing of the DNA strands during the replication process, and are broadly classified as type I or II according to their ability to cleave one or both strands, though sub-classes are now recognized. Camptothecin (see page 384) is an inhibitor of topoisomerase I. Etoposide is believed to inhibit strand-rejoining ability by stabilizing the topoisomerase II–DNA complex in a cleavage state, leading to double strand breaks and cell death. Rather than simply inhibiting the enzyme, etoposide converts topoisomerase II into a potent cellular toxin, and the term topoisomerase II poison is now often employed to distinguish the effect from an agent that merely alters catalytic activity. Development of other topoisomerase inhibitors based on podophyllotoxin-related lignans is an active area of research. Biological activity in this series of compounds is very dependent on the presence of the trans-fused five-membered lactone ring, this type of fusion producing a highly strained system. Ring strain is markedly reduced in the corresponding cis-fused system, and the natural compounds are easily and rapidly converted into these cis-fused lactones by treatment with very mild bases, via enol tautomers or enolate anions (Figure 4.23). Picropodophyllin is almost devoid of cytotoxic properties.

base removes acidic proton α to carbonyl and generates enolate anion

OH O

re-formation of keto form results in change of stereochemistry

OH

O

O O

O

O

O NaOAc

H

OH

O

O

O

H

HB O

O

B MeO

OMe OMe

podophyllotoxin Figure 4.23

MeO

OMe OMe

MeO

OMe OMe

picropodophyllin

156 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 4.3 (continued) Podophyllotoxin is also found in significant amounts in the roots of other Podophyllum species, and in closely related genera such as Diphylleia (Berberidaceae). Fungi isolated from the roots of Podophyllum species have also been found to produce podophyllotoxin in culture, suggesting possible gene transfer between the plant and the endophytic fungus. These fungi include Trametes hirsuta from rhizomes of Podophyllum hexandrum, and Phialocephala fortinii from Podophyllum peltatum. Yields tend to be rather low, however, and would need considerable improvement to offer an alternative source to the plant material.

Phenylpropenes The reductive sequence from an appropriate cinnamic acid to the corresponding cinnamyl alcohol is utilized for the production of various phenylpropene derivatives. Thus cinnamaldehyde (Figure 4.24) is the principal component in the oil from the bark of cinnamon (Cinnamomum zeylanicum; Lauraceae), widely used as a spice and flavouring. Fresh bark is known to contain high levels of the ester cinnamyl acetate, and cinnamaldehyde is released from this by fermentation processes which are part of commercial preparation of the bark, presumably by enzymic hydrolysis and participation of the reversible alcohol dehydrogenase. Cinnamon leaf, on the other hand, contains large amounts of eugenol (Figure 4.24) and much smaller amounts of cinnamaldehyde. Eugenol is also the principal constituent in oil from cloves (Syzygium aromaticum; Myrtaceae), used for many years as a dental anaesthetic as well as for flavouring. Allylphenols (e.g. eugenol) or propenylphenols (e.g. isoeugenol) both originate from appropriate cinnamyl alcohols by way of acetate esters (Figure 4.25), though the two groups of compounds differ with respect to the position of the side-chain double bond. The acetate function provides a leaving group, loss of which is facilitated by the presence of the para hydroxyl group on the aromatic ring, leading to a quinonemethide intermediate. This can

be reduced by addition of hydride (from NADPH) generating the different side-chains according to the position of attack; the genes and enzymes involved in the two processes have been characterized. Myristicin (Figure 4.24) from nutmeg (Myristica fragrans; Myristicaceae) is a further example of an allylphenol found in flavouring materials. Myristicin also has a history of being employed as a mild hallucinogen via ingestion of ground nutmeg. It is probably metabolized in the body via an amination reaction to give an amfetamine-like derivative (see page 404). Anethole is the main component in oils from aniseed (Pimpinella anisum; Umbelliferae/Apiaceae), star anise (Illicium verum; Illiciaceae) and fennel (Foeniculum vulgare; Umbelliferae/Apiaceae). The presence of propenyl components in flavouring materials such as cinnamon, star anise, nutmeg, and sassafras (Sassafras albidum; Lauraceae) has reduced their commercial use somewhat, since these constituents have been shown to be weak carcinogens in laboratory tests on animals. In the case of safrole (Figure 4.26), the main component of sassafras oil, this toxicity has been shown to arise from hydroxylation in the side-chain followed by sulfation, giving an agent which binds to cellular macromolecules. Further data on volatile oils containing aromatic constituents isolated from these and other plant materials are given in Table 4.1. Volatile oils in which the main components are terpenoid in nature are listed in Table 5.1, page 158.

OCOCH3

CHO

MeO cinnamaldehyde

Figure 4.24

cinnamyl acetate

OMe

OMe

anethole

estragole (methylchavicol)

MeO OH eugenol

O O myristicin

OMe

MeO OMe elemicin

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 157 attack of H hydride at g-position

loss of acetate leaving group; formation of quinonemethide O OH

esterification

O

Me

Me O

O

OMe

OMe NADPH

CH3COSCoA

E1

≡ OMe

OMe

OMe

OH

OH

OH

coniferyl alcohol

OH isoeugenol (propenylphenol)

OH

NADPH

attack of hydride at a-position

E2

H

coniferyl acetate

OMe

E1: isoeugenol synthase 1 E2: eugenol synthase 1 E3: eugenol O-methyltransferase

OH

SAM OMe OH eugenol (allylphenol)

OMe

E3 OMe

Figure 4.25

HO

O

HO3SO

O O safrole

O O

O safrole 1′-sulfate

Figure 4.26

Benzoic Acids from C6 C3 Compounds Benzoic acids are structurally simple natural products, so it is quite surprising just how many different pathways may be employed for their biosynthesis. Pathways to the same compound may be quite different according to the organism, and sometimes more than one pathway may exist in a single organism. Some of the simple hydroxybenzoic acids (C6 C1 compounds), such as gallic acid and 4-hydroxybenzoic acid, can be formed directly from intermediates early in the shikimate pathway, e.g. 3-dehydroshikimic acid (see page 138) or chorismic acid (see page 141), but alternative routes exist in which cinnamic acid derivatives (C6 C3 compounds) are cleaved at the double bond and lose two carbon atoms from the side-chain. Thus, 4-coumaric acid may act as a precursor of 4-hydroxybenzoic acid, and ferulic acid may

give vanillic acid (4-hydroxy-3-methoxybenzoic acid) (Figure 4.27). Alternatively, cinnamic acid itself may be converted into benzoic acid. A sequence analogous to that involved in the β-oxidation of fatty acids (see page 18) is possible, so that the double bond in the coenzyme A ester would be hydrated, the hydroxyl group oxidized to a ketone, and the β-ketoester would then lose acetyl-CoA by a reverse Claisen reaction, giving the coenzyme A ester of 4-hydroxybenzoic acid. Whilst this sequence has been generally accepted, there is also evidence to support another side-chain cleavage mechanism which is different from the fatty acid β-oxidation pathway (Figure 4.27). Coenzyme A esters are not involved, and though a similar hydration of the double bond occurs, chain shortening features a reverse aldol reaction, generating the appropriate aromatic aldehyde. The corresponding acid is then formed via an NAD+ -dependent oxidation step. Thus, aromatic aldehydes such as vanillin, the main flavour compound in vanilla (pods of the orchid Vanilla planiflora; Orchidaceae), would be formed from the correspondingly substituted cinnamic acid without proceeding through intermediate benzoic acids or esters. In some plants and microorganisms, yet another variant may be implicated. This resembles the latter route in employing a reverse aldol cleavage, but utilizes CoA esters and bifunctional hydratase/lyase enzymes (Figure 4.27). Whilst the substitution pattern in these C6 C1 derivatives is generally built up at the C6 C3 cinnamic acid stage, there exists the possibility of further hydroxylations and/or methylations occurring at the C6 C1 level prior to chain

Plant source

Pimpinella anisum (Umbelliferae/Apiaceae) Illicium verum (Illiciaceae)

Cinnamomum cassia (Lauraceae)

Cinnamomum zeylanicum (Lauraceae)

Cinnamomum zeylanicum (Lauraceae)

Syzygium aromaticum (Eugenia caryophyllus) (Myrtaceae)

Foeniculum vulgare (Umbelliferae/Apiaceae)

Oil

Aniseed (Anise) Star anise

Cassia

Cinnamon bark

Cinnamon leaf

Clove

Fennel

ripe fruit

dried flower buds

leaf

2–5

15–20

0.5–0.7

1–2

1–2

5–8

ripe fruit

dried bark, or leaves and twigs dried bark

2–3

Oil content %

ripe fruit

Plant part used

Table 4.1 Volatile oils (i): containing principally aromatic compounds

anethole (50–70) fenchone (10–20) estragole (3–20)

eugenol (75–90) eugenyl acetate (10–15) β-caryophyllene (3)

cinnamaldehyde (70–80) eugenol (1–13) cinnamyl acetate (3–4) eugenol (70–95)

cinnamaldehyde (70–90) 2-methoxycinnamaldehyde (12)

anethole (80–90) estragole (1–6) anethole (80–90) estragole (1–6)

Major constituents with typical (%) composition

flavour, carminative, aromatherapy

flavour, aromatherapy, antiseptic

flavour

flavour, carminative, aromatherapy

flavour, carminative; fruits contain substantial amounts of shikimic (about 3%) and quinic acids flavour, carminative; known as cinnamon oil in USA

flavour, carminative, aromatherapy

Uses, notes

158 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Gaultheria procumbens (Ericacae) or Betula lenta (Betulaceae)

Wintergreen 0.7–1.5

0.2–0.6

bark

5–16

leaves

seed

sabinene (17–28) α-pinene (14–22) β-pinene (9–15) terpinen-4-ol (6–9) myristicin (4–8) elemicin (2) methyl salicylate (98)

flavour, antiseptic, antirheumatic; prior to distillation, plant material is macerated with water to allow enzymic hydrolysis of glycosides; methyl salicylate is now produced synthetically

flavour, carminative, aromatherapy; although the main constituents are terpenoids, most of the flavour comes from the minor aromatic constituents, myristicin, elemicin, etc.; myristicin is hallucinogenic (see page 156)

less harsh techniques, such as expression or solvent extraction, may be employed. These oils, which typically contain a complex mixture of low boiling-point components, are widely used in flavouring, perfumery, and aromatherapy. Only a small number of oils have useful therapeutic properties, e.g. clove and dill, though a wide range of oils is now exploited for aromatherapy. Most of those employed in medicines are simply added for flavouring purposes. Some of the materials are commercially important as sources of chemicals used industrially, e.g. turpentine. b For convenience, the major oils listed are divided into two groups. Those oils which contain principally chemicals that are aromatic in nature and which are derived by the shikimate pathway are given in this table. Oils which are composed predominantly of terpenoid compounds are listed in Table 5.1 on page 200, since they are derived via the methylerythritol phosphate or mevalonate pathways. It must be appreciated that many oils may contain aromatic and terpenoid components, but usually one group predominates. The oil yields and the exact composition of any sample of oil will be variable, depending on the particular plant material used in its preparation. The quality of an oil and its commercial value is dependent on the proportion of the various components. Structures of aromatics are shown in Figure 4.23 (see page 156).

a Volatile or essential oils are usually obtained from the appropriate plant material by steam distillation. If certain components are unstable at these temperatures, however, then other,

Notes:

Myristica fragrans (Myristicaceae)

Nutmeg

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 159

160 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

CO2H

COSCoA

COSCoA

COSCoA

HO HSCoA ATP E1

R

NAD+

H2 O E2 E6

R

OH R = H, 4-coumaric acid R = OMe, ferulic acid

COSCoA

E4

HSCoA

COSCoA

CO2H NAD+ E7

R OH R = H, 4-hydroxybenzaldehyde R = OMe, vanillin

E1: CoA ligase E2: enoyl-CoA hydratase E3: dehydrogenase

CH3COSCoA

O

CHO

reverse aldol

OH

reverse Claisen

E6

HO

R

OH HSCoA

CO2H

b-oxidation pathway, as in fatty acid metabolism (Figure 2.11)

R

OH

reverse aldol

HO

CO2H

E3

R

OH R = H, 4-coumaroyl-CoA R = OMe, feruloyl-CoA

H2O

O

E4: thiolase E5: thioesterase

E5

R

R OH

OH R = H, 4-hydroxybenzoic acid R = OMe, vanillic acid E6: enoyl-CoA hydratase/lyase E7: dehydrogenase

Figure 4.27

hydroxylation

CO2H

O2 NADPH

methylation

CO2H OH

CO2Me

SAM

E1

CO2H

OH

OCOCH3

E2

benzoic acid

salicylic acid

aspirin (acetylsalicylic acid)

methyl salicylate

side-chain cleavage CO2H

CO2H

hydroxylation OH

cinnamic acid

side-chain cleavage

2-coumaric acid

E1: benzoic acid 2-hydroxylase E2: salicylic acid carboxyl methyltransferase

Figure 4.28

glucosylation

CHO OH

salicylaldehyde

UDPGlc

reduction

CHO

CH2OH

OGlc

OGlc

salicin

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 161 shortening, and this is known in certain examples. Salicylic acid (Figure 4.28) is synthesized in microorganisms directly from isochorismic acid (see page 141), but can arise in plants by two other mechanisms. It can be produced by hydroxylation of benzoic acid, or by side-chain cleavage of 2-coumaric acid, which itself is formed by an ortho-hydroxylation of cinnamic acid. Methyl salicylate is the principal component of oil of wintergreen from Gaultheria procumbens (Ericaceae), used for many years for pain relief. It is derived by SAM-dependent methylation of salicylic acid. The salicyl alcohol derivative salicin, found in many species of willow (Salix species; Salicaceae), is not derived from salicylic acid, but probably via glucosylation of salicylaldehyde and then reduction of the carbonyl (Figure 4.28). Salicin is responsible for the analgesic and antipyretic effects of willow barks, widely used for centuries. It provided the template for synthesis of acetylsalicylic acid (aspirin; Figure 4.28), a more effective and widely used pain-killer.

Coumarins The hydroxylation of cinnamic acids ortho to the side-chain as seen in the biosynthesis of salicylic acid (Figure 4.28) is a crucial step in the formation of a group of cinnamic acid lactone derivatives, the coumarins. Whilst the direct hydroxylation of the aromatic ring of the cinnamic acids is common, hydroxylation generally involves initially the 4-position para to the side-chain, and CO2H

CO2H E2

OH 2-coumaric acid

cinnamic acid O2 E1 NADPH

E2

HO

4-coumaric acid

CO2H

trans–cis isomerization

HO

OH

lactone formation

OH

CO2H

HO

2,4-dihydroxycinnamic acid MeO GlcO

E1: cinnamate 4-hydroxylase E2: cinnamate/coumarate 2-hydroxylase

Figure 4.29

OH

O coumarin

O

CO2H

CO2H HO

subsequent hydroxylations then proceed ortho to this substituent (see page 149). In contrast, for the coumarins, hydroxylation of cinnamic acid or 4-coumaric acid can occur ortho to the side-chain (Figure 4.29). In the latter case, the 2,4-dihydroxycinnamic acid produced confusingly seems to possess the meta hydroxylation pattern characteristic of phenols derived via the acetate pathway. Recognition of the C6 C3 skeleton should help to avoid this confusion. The two 2-hydroxycinnamic acids then suffer a change in configuration in the side-chain, from the trans (E) to the less stable cis (Z) form. Whilst trans–cis isomerization would be unfavourable in the case of a single isolated double bond, the fully conjugated system in the cinnamic acids allows this process to occur quite readily, and UV irradiation, e.g. daylight, is sufficient to produce equilibrium mixtures which can be separated. The absorption of energy promotes an electron from the π-orbital to a higher energy state, i.e. the π∗ -orbital, thus temporarily destroying the double-bond character and allowing rotation. Loss of the absorbed energy then results in re-formation of the double bond, but in the cis-configuration. In conjugated systems, the π–π∗ energy difference is considerably less than with a non-conjugated double bond. Chemical lactonization can occur on treatment with acid. Both the trans–cis isomerization and the lactonization are enzyme mediated in nature, and light is not necessary for coumarin biosynthesis. However, the enzyme activities are poorly characterized. Cinnamic acid and 4-coumaric acid give rise to the coumarins coumarin and umbelliferone respectively (Figure 4.29). Other coumarins with

MeO O scopolin

O

O

O

umbelliferone

HO

HO O scopoletin

O

HO

O aesculetin

O

162 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition CO2H OGlc (E)-2-coumaric acid glucoside enzymic hydrolysis O

CO2H

O

Glc (Z)-2-coumaric acid glucoside

O

coumarin

Figure 4.30

additional oxygen substituents on the aromatic ring, e.g. aesculetin (esculetin) and scopoletin, appear to be derived by modification of umbelliferone, rather than by a general cinnamic acid to coumarin pathway. This indicates that the hydroxylation meta to the existing hydroxyl, discussed above, is a rather uncommon occurrence and restricted to certain substrates. Coumarins are widely distributed in plants, both in the free form and as glycosides, and are commonly found in families such as the Umbelliferae/Apiaceae and Rutaceae. Coumarin itself is found in sweet clover (Melilotus alba; Leguminosae/Fabaceae) and contributes to the smell of new-mown hay, though there is evidence that the plants actually contain the glucosides of (E)- and

O COSCoA

lactone formation and enolization

(Z)-2-coumaric acid (Figure 4.30), and coumarin is only liberated as a result of enzymic hydrolysis and lactonization through damage to the plant tissues during harvesting and processing (Figure 4.30). If sweet clover is allowed to ferment, then 4-hydroxycoumarin is produced by the action of microorganisms on 2-coumaric acid (Figure 4.31). This can then react with formaldehyde, which is usually present from microbial degradative reactions, combining to give dicoumarol. Dicoumarol is a compound with pronounced blood anticoagulant properties which can cause internal bleeding and death of livestock, and is the forerunner of the warfarin group of rodenticides and medicinal anticoagulants [Box 4.4]. Many other natural coumarins have a more complex carbon framework and incorporate extra carbon atoms derived from an isoprene unit (Figure 4.33). For these compounds, there is considerably more evidence available from enzymic and genetic studies than for the simple coumarins. The aromatic ring in umbelliferone is activated at positions ortho to the hydroxyl group and can be alkylated by a suitable alkylating agent, in this case dimethylallyl diphosphate. The newly introduced dimethylallyl group in demethylsuberosin is then able to cyclize with the phenol group to give marmesin. This transformation is catalysed by a cytochrome P-450-dependent monooxygenase and requires cofactors NADPH and molecular oxygen. For many years, the cyclization had been postulated to involve an intermediate epoxide, so that nucleophilic attack of the phenol onto the epoxide group might lead to formation of either five-membered furan

OH

O

dehydration follows

OH

H O

O O 4-hydroxycoumarin

OH

O

OH

O O dicoumarol

O

O

– H2 O

HCHO

Figure 4.31

OH

COSCoA H

OH

H

aldol reaction OH

H

nucleophilic attack onto the enone system: Michael reaction OH

O

O

O

O

O

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 163

Box 4.4 Dicoumarol and Warfarin The cause of fatal haemorrhages in animals fed spoiled sweet clover (Melilotus officinalis; Leguminosae/Fabaceae) was traced to dicoumarol (bishydroxycoumarin; Figure 4.31). This agent interferes with the effects of vitamin K in blood coagulation (see page 183), the blood loses its ability to clot, and thus minor injuries can lead to severe internal bleeding. Synthetic dicoumarol has been used as an oral blood anticoagulant in the treatment of thrombosis, where the risk of blood clots becomes life threatening. It has been superseded by salts of warfarin and in some cases the nitro analogue acenocoumarol (nicoumalone; Figure 4.32), which are synthetic developments from the natural product. An overdose of warfarin may be countered by injection of vitamin K1 . Warfarin was initially developed as a rodenticide and has been widely employed for many years as the first-choice agent, particularly for destruction of rats. After consumption of warfarin-treated bait, rats die from internal haemorrhage. Other coumarin derivatives employed as rodenticides include coumachlor and coumatetralyl (Figure 4.32). In an increasing number of cases, rodents are developing resistance towards warfarin, an ability which has been traced to elevated production of vitamin K by their intestinal microflora. Modified structures difenacoum, brodifacoum, bromadiolone, and flocoumafen have been found to be more potent than warfarin, and are also effective against rodents that have become resistant to warfarin.

NO2

OH

Cl

O

O

O RS

RS

RS

RS acenocoumarol (nicoumalone)

O O warfarin

coumachlor

RS

RS

coumatetralyl

OH RS

RS RS

RS

O R = H, difenacoum R = Br, brodifacoum

R

bromadiolone

Br

flocoumafen

CF3

Figure 4.32

or six-membered pyran heterocycles, as commonly encountered in natural products (Figure 4.34). Although the reactions of Figure 4.34 offer a convenient rationalization for cyclization, epoxide intermediates have not been demonstrated in any of the enzymic systems so far investigated; therefore, some direct oxidative cyclization mechanism must operate. A second cytochrome P-450-dependent monooxygenase enzyme then cleaves off the hydroxyisopropyl fragment (as acetone) from marmesin to give the furocoumarin psoralen. This does not involve any

hydroxylated intermediate, and cleavage is believed to be initiated by a radical abstraction process (Figure 4.35). Psoralen can act as a precursor for the further substituted furocoumarins bergapten, xanthotoxin, and isopimpinellin (Figure 4.33), such modifications occurring late in the biosynthetic sequence rather than at the cinnamic acid stage. These transformations are initiated by yet further cytochrome P-450-dependent enzymes, introducing hydroxyls into the aromatic ring. Psoralen, bergapten, and related compounds are termed ‘linear’ furocoumarins.

164 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition C-alkylation at activated position ortho to phenol

OPP

5

3

DMAPP HO

O2 NADPH

4

6

O

8

O

E1

HO

umbelliferone

O

O

E3

OH

O

E5

O

O

E4

O

hydroxylation methylation

O

O

O

O

O

O

OMe

OMe

OH

isopimpinellin

xanthotoxin

xanthotoxol

E1: umbelliferone 6-prenyltransferase E2: marmesin synthase E3: psoralen synthase

O2 NADPH

O

O

psoralen (linear furocoumarin)

E6

E7

O

O

O2 NADPH

SAM O

O

O2 NADPH

O bergaptol

bergapten

O (+)-marmesin

SAM

OMe

O

demethylsuberosin

OMe

O

HO

E2

E4: psoralen 5-monooxygenase E5: bergaptol O-methyltransferase

O

O

O

O

angelicin (angular furocoumarin)

E6: psoralen 8-monooxygenase E7: xanthotoxol O-methyltransferase

Figure 4.33 H O – H2O HO O

HO nucleophilic attack onto protonated epoxide

H O

O

5-membered furan ring

HO

– H2O O

HO

O

6-membered pyran ring

Figure 4.34 Enz

Fe O

oxidation leading to radical H

H

cleavage of sidechain carbons

HO O marmesin

E1: psoralen synthase

Figure 4.35

O

E1

Fe OH

O2 NADPH

HO O

Enz

HO O

side-chain carbons released as acetone

O

O

O – H 2O O

HO

OH

O psoralen

O

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 165 ‘Angular’ furocoumarins, e.g. angelicin (Figure 4.33), can arise by a similar sequence of reactions, but these involve initial dimethylallylation at the alternative position ortho to the phenol. An isoprene-derived furan ring system has already been noted in the formation of khellin (see page 112), though the aromatic ring to which it was

fused was in that case a product of the acetate pathway. Linear furocoumarins (psoralens) can be troublesome to humans since they can cause photosensitization towards UV light, resulting in sunburn or serious blistering. Used medicinally, this effect may be valuable in promoting skin pigmentation and treating psoriasis [Box 4.5].

Box 4.5 Psoralens Psoralens are linear furocoumarins which are widely distributed in plants, but are particularly abundant in the Umbelliferae/Apiaceae and Rutaceae. The most common examples are psoralen, bergapten, xanthotoxin, and isopimpinellin (Figure 4.33). Plants containing psoralens have been used internally and externally to promote skin pigmentation and suntanning. Bergamot oil obtained from the peel of Citrus aurantium ssp. bergamia (Rutaceae) (see page 165) can contain up to 5% bergapten and is frequently used in external suntan preparations. The psoralen, because of its extended chromophore, absorbs in the near UV and allows this radiation to stimulate formation of melanin pigments (see page 147). Methoxsalen (xanthotoxin; 8-methoxypsoralen; Figure 4.36), a constituent of the fruits of Ammi majus (Umbelliferae/Apiaceae), is used medically to facilitate skin repigmentation where severe blemishes exist (vitiligo). An oral dose of methoxsalen is followed by long-wave UV irradiation, though such treatments must be very carefully regulated to minimize the risk of burning, cataract formation, and the possibility of causing skin cancer. The treatment is often referred to as PUVA (psoralen + UV-A). PUVA is also of value in the treatment of psoriasis, a widespread condition characterized by proliferation of skin cells. Similarly, methoxsalen is taken orally, prior to UV treatment. Reaction with psoralens inhibits DNA replication and reduces the rate of cell division. Because of their planar nature, psoralens intercalate into DNA, and this enables a UV-initiated cycloaddition reaction between pyrimidine bases (primarily thymine) in DNA and the furan ring of psoralens (Figure 4.36). A second cycloaddition can then occur, this time involving the pyrone ring, leading to interstrand cross-linking of the nucleic acid.

O

O

HN O

O O

HN O

N

thymine in DNA

O

OMe

xanthotoxin (methoxsalen)

N

O

O hν

N

O

O

OMe

psoralen–DNA adduct

N

O

O O

HN O

HN

O

hν

O

HN O

N

O

OMe

psoralen–DNA interstrand cross-link

Figure 4.36 A troublesome extension of these effects can arise from the handling of plants which contain significant levels of furocoumarins. Celery (Apium graveolens; Umbelliferae/Apiaceae) is normally free of such compounds, but fungal infection with the natural parasite Sclerotinia sclerotiorum induces the synthesis of furocoumarins (xanthotoxin and others) as a response to the infection. Field workers handling these infected plants may become very sensitive to UV light and suffer from a form of sunburn termed photophytodermatitis. Infected parsley (Petroselinum crispum) can give similar effects. Handling of rue (Ruta graveolens; Rutaceae) or giant hogweed (Heracleum mantegazzianum; Umbelliferae/Apiaceae), which naturally contain significant amounts of psoralen, bergapten, and xanthotoxin, can cause similar unpleasant reactions, or more commonly rapid blistering by direct contact with the sap. The giant hogweed can be particularly dangerous. Individuals vary in their sensitivity towards furocoumarins; some are unaffected, whilst others tend to become sensitized by an initial exposure and then develop the allergic response on subsequent exposures.

166 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition chain extension; acetate pathway with a cinnamoyl-CoA starter group O

O

OH di-enol tautomer

2 × malonyl-CoA SCoA

O

HO

O SCoA

OH

≡

HO

O SCoA

HO

4-hydroxycinnamoyl-CoA

lactone formation

OMe

OH

SAM SAM O MeO

O

O

O

HO yangonin

bisnoryangonin

Figure 4.37 AROMATIC POLYKETIDES Cinnamic acids, as their coenzyme A esters, may also function as starter units for chain extension with malonyl-CoA units via PKSs, thus combining elements of the shikimate and acetate pathways (see page 116). Most commonly, three C2 units are added via malonate, giving rise to flavonoids and stilbenes (page 116). Products formed from a cinnamoyl-CoA starter plus one or two C2 units from malonyl-CoA are rarer, but provide excellent examples to complete the broader picture. Styrylpyrones, Diarylheptanoids Styrylpyrones are formed from a cinnamoyl-CoA and two malonyl-CoA extender units. Short poly-β-keto chains

frequently cyclize to form a lactone derivative (compare triacetic acid lactone, page 101). Thus, Figure 4.37 shows the proposed derivation of yangonin via cyclization of the di-enol tautomer of the polyketide formed from 4-hydroxycinnamoyl-CoA. Two methylation reactions complete the sequence. Indeed, the suggested intermediate bisnoryangonin is often produced in small amounts as a derailment product when recombinant chalcone synthase enzymes (see page 169) are incubated with 4-hydroxycinnamoyl-CoA and malonyl-CoA, being released from the enzyme after only two instead of three malonate condensations. Yangonin and a series of related structures form the active principles of kava root (Piper methysticum; Piperaceae), a herbal remedy popular for its anxiolytic activity [Box 4.6].

Box 4.6 Kava Aqueous extracts from the root and rhizome of Piper methysticum (Piperaceae) have long been consumed as an intoxicating beverage by the peoples of Pacific islands comprising Polynesia, Melanesia, and Micronesia, and the name kava or kava-kava referred to this drink. In herbal medicine, the dried root and rhizome is now described as kava, and it is used for the treatment of anxiety, nervous tension, agitation, and insomnia. The pharmacological activity is associated with a group of styrylpyrone derivatives termed kavalactones, good-quality roots containing 5–8% kavalactones. At least 18 kavalactones have been characterized, the six major ones being four enolides kawain, methysticin and their corresponding dihydro derivatives, and the two dienolides yangonin and demethoxyyangonin (Figure 4.38). The enolides have a reduced pyrone ring and a chiral centre. Clinical trials have indicated kava extracts to be effective as an anxiolytic, the kavalactones also displaying anticonvulsive, analgesic, and central muscle-relaxing action. Several of these compounds have been shown to have an effect on neurotransmitter systems, including those involving glutamate, GABA, dopamine, and serotonin. The safety of kava products has been questioned following hepatotoxic side-effects: severe liver damage in patients taking kava extracts has been reported. It is suggested that the traditional usage of aqueous extracts appears to produce little or no hepatotoxicity, and the problems stem from using commercial organic solvent extracts. Water extracts also contain glutathione (see page 125), which appears to provide protection against toxicity through reaction with the lactone ring. A number of human cytochrome P-450-dependent enzymes are also known to be inhibited by some of the kavalactones.

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 167

Box 4.6 (continued) OMe

H

O

OMe

OMe

O

H

O

O

O

H

O

OMe

O

O

H

O kawain

O

O

dihydrokawain

methysticin

dihydromethysticin

OMe

O

O

OMe

O

O

O

MeO yangonin

demethoxyyangonin

Figure 4.38

Chain extension with a single malonyl-CoA unit is exemplified by raspberry ketone (Figure 4.39), the aroma constituent of ripe raspberry fruits (Rubus idaeus; Rosaceae). However, it is immediately apparent that the side-chain in this product is actually one carbon shorter than predicted; this PKS also catalyses hydrolysis and decarboxylation. A subsequent reduction generates raspberry ketone. Piperoyl-CoA is implicated in the biosynthesis of the alkaloid piperine (see page 328); this retains the two carbon atoms from the malonate extender.

Characteristic constituents of turmeric (Curcuma longa; Zingiberaceae), an essential spice ingredient in curries, are a group of curcuminoids, diarylheptanoids responsible for the yellow colour. The principle component, curcumin (Figure 4.40), is now attracting considerable interest because of its anti-inflammatory, antiulcer, antitumour, and cancer-preventative properties. Curcuminoids are produced by a type III PKS-like enzyme (see page 68); and as with the raspberry ketone example, a single extra carbon is incorporated from malonate. In this

chain extension with one malonyl-CoA unit O

O

malonyl-CoA

O

SCoA

O O

SCoA

HO

SCoA

O

HO

piperoyl-CoA

O

O

malonyl-CoA

NADPH

SCoA HO

E1: benzalacetone synthase E2: benzalacetone reductase

Figure 4.39

O

E1 CO2 HSCoA

HO 4-hydroxybenzalacetone

E2

HO raspberry ketone 4-hydroxyphenylbutan-2-one

168 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O MeO

E1

O

SCoA

HO E1

O O

O

SCoA

O

MeO

MeO HO

Claisen reaction with decarboxylation

reduction O

chain extension with one malonyl-CoA unit

O

MeO

SCoA

HO

O

malonyl-CoA

O

HO

MeO

OMe OH

E1 O

HO

6-gingerol

Claisen reaction with decarboxylation

O CoAS

OH

H

O

MeO

OMe

− H2O O

HO

OH

MeO O

OH

MeO

HO

OMe

6-shogaol HO E1: curcuminoid synthase

OH curcumin curcumin exists as a ketoenol rather than diketo structure

Figure 4.40

series of compounds, a further cinnamoyl group is also added. It seems likely that this unit is incorporated via a Claisen reaction with the β-ketoacid formed by hydrolysis of this β-keto ester. Decarboxylation is part of the Claisen process as with normal malonate chain extension. Related to the curcuminoids, and in some plants often co-occurring with them, are compounds such as the gingerols and shogaols. These are presumably derived by a variant of the curcuminoid pathway that utilizes a fatty acyl thioester rather than a cinnamoyl thioester in the further Claisen reaction (Figure 4.40). Chain lengths in this fatty acyl group vary; thus, 6-gingerol has incorporated a six-carbon fragment, whilst other analogues, e.g. 8-gingerol and 10-gingerol, employ 8- or 10-carbon fatty acid esters. 6-Gingerol is a major pungent component in ginger root (Zingiber officinale; Zingiberaceae), widely employed as a flavouring and spice; the distinctive aroma of ginger arises from volatile sesquiterpenes (see page 211). Shogaols are dehydrated analogues of gingerols, and tend to be the predominant pungent agents in dried root,

so may be formed during the drying process. Ginger has useful anti-inflammatory activity due to the gingerols. Flavonoids and Stilbenes Flavonoids and stilbenes are products from a cinnamoyl-CoA starter unit, with chain extension using three molecules of malonyl-CoA. Reactions are catalysed by a type III PKS enzyme (see page 68). These enzymes do not utilize ACPs, but instead employ coenzyme A esters and have a single active site to perform the necessary series of reactions, e.g. chain extension, condensation, cyclization. Chain extension of 4-hydroxycinnamoyl-CoA with three molecules of malonyl-CoA gives initially a polyketide (Figure 4.41) which can be folded in two different ways. These allow aldol or Claisen-like reactions to occur, generating aromatic rings, as already seen in Chapter 3 (see page 100). Stilbenes (e.g. resveratrol) or chalcones (e.g. naringenin-chalcone) are the end-products formed

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 169 chain extension; acetate pathway with a cinnamoyl-CoA starter group OH

OH

3 × malonyl-CoA

CoAS

CoAS O

O

O

E1 E2

O

O 4-hydroxycinnamoyl-CoA

different folding modes OH

OH O

O

O

OH OH

E2

O OH

Claisen

O E1

Claisen

OH O

O

O decarboxylation dehydration enolization OH

O E1

CO2H O

SCoA

E3 O

aldol + ester hydrolysis

E2

CO2

O NADPH

SCoA O

O

SCoA O

O

OH

NADPH

O

E3

H

enolization

(E1)

OH

OH

O

H OH O

O

(E1) HO

OH

HO

dehydration enolization OH

OH

HO OH O O naringenin-chalcone isoliquiritigenin Michael-type nucleophilic (chalcone) (chalcone) attack of OH onto a,b-unsaturated ketone

OH resveratrol (stilbene)

E4

E4 E1: chalcone synthase (naringenin-chalcone synthase) E2: stilbene synthase (resveratrol synthase) E3: chalcone reductase E4: chalcone isomerase

OH HO

O

OH HO

OH O naringenin (flavanone)

O

O liquiritigenin (flavanone)

Figure 4.41 by enzymes stilbene synthase and chalcone synthase respectively. Both structures nicely illustrate the different characteristic oxygenation patterns in the two aromatic rings derived from the acetate or shikimate pathways. Though cyclizing the same substrate in different ways, chalcone synthase and stilbene synthase enzymes are

found to share some 75–90% amino acid sequence identity. Whilst chalcone synthase enzymes appear to be found in all plants, stilbene synthase activity has a much more restricted distribution. With the stilbenes, the terminal ester function is no longer present; therefore, hydrolysis and decarboxylation have also taken place

170 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition during this transformation. Carboxylated stilbenes are rare in nature and are not intermediates in the stilbene synthase reaction. Mutagenesis studies have confirmed that hydrolysis of the thioester bond and decarboxylation precede aromatization by the necessary dehydration/enolization reactions. Indeed, it appears that it is the thioesterase activity component of the stilbene synthase enzyme that is primarily responsible for the stilbene-type cyclization through an aldol reaction rather than the chalcone-type cyclization via a Claisen reaction. Through appropriate amino acid replacements it is possible to convert chalcone synthase into proteins with stilbene synthase activity. The stilbene resveratrol has assumed considerable relevance in recent years as a constituent of grapes and wine, as well as other food products, with antioxidant, anti-inflammatory, inhibition of platelet aggregation, and protective activity against cancer and cardiovascular diseases. Coupled with the cardiovascular benefits of moderate amounts of alcohol, and the beneficial antioxidant effects of flavonoids (see page 171), red wine has now emerged as an unlikely but most acceptable medicinal agent. Resveratrol is available as a dietary supplement, though it is not currently regarded as a therapeutic agent. Commercial material is isolated from grapeskins or Japanese knotweed (Polygonum cuspidatum, syn. Fallopia japonica; Polygonaceae). Resveratrol also has antifungal properties and is formed as a phytoalexin in some plant species, including grapes and peanuts, as a result of infection. It is now feasible to increase resistance to fungi by expressing a stilbene synthase gene in a plant that normally does not produce resveratrol. The precursors normally destined for chalcone biosynthesis are thus channelled into stilbene synthesis. Another natural stilbene of medicinal interest is combretastatin A-4 (Figure 4.42), isolated from bark of the tree Combreta caffrum (Solanaceae). Apart from the substitution pattern, this compound differs from resveratrol in that the double bond has the Z-configuration. Combretastatin A-4 is a potent cytotoxic agent which strongly inhibits polymerization of tubulin by binding to the colchicine site (see page 361); the structural similarity to colchicine is readily appreciated (Figure 4.42). The water-soluble phosphate pro-drug is currently showing considerable promise in clinical trials against various solid tumours; it appears to reduce blood flow in the tumour markedly. Chalcones act as precursors for a vast range of flavonoid derivatives found throughout the plant kingdom. Enzymic and genetic studies for this group of compounds are now well advanced. Most flavonoids contain a six-membered heterocyclic ring, formed by Michael-type nucleophilic attack of a phenol group onto the unsaturated

MeO MeO OMe

OR OMe

R = H, combretastatin A-4 R = P, combretastatin A-4 phosphate MeO NHAc H

MeO MeO

O OMe colchicine

Figure 4.42 ketone to give a flavanone, e.g. naringenin (Figure 4.41). This isomerization can occur chemically, with acid conditions favouring the flavanone and basic conditions the chalcone. In nature, however, the reaction is enzyme catalysed and stereospecific, resulting in formation of a single flavanone enantiomer. Many flavonoid structures, e.g. liquiritigenin, have lost one of the hydroxyl groups, so that the acetate-derived aromatic ring has a resorcinol oxygenation pattern rather than the phloroglucinol system. This modification has been tracked down to the action of a chalcone reductase enzyme concomitant with the chalcone synthase; thus, isoliquiritigenin is produced rather than naringenin-chalcone. Reduction of the linear poly-β-ketoester or of the cyclic trione might be formulated; evidence points to the cyclic trione as substrate. The last stage(s) in chalcone formation through enolization or dehydration/enolization are probably spontaneous and non-enzymic. Flavanones can then give rise to many variants on this basic skeleton, e.g. flavones, flavonols, anthocyanidins, and catechins (Figure 4.43). Most of the enzyme systems show rather broad substrate specificity and convert compounds with different oxygenation patterns in the aryl substituent. Modifications to the hydroxylation patterns in either of the two aromatic rings may occur, generally at the flavanone or dihydroflavonol stage, and methylation, glycosylation, and dimethylallylation are also possible, increasing the range of compounds enormously. A high proportion of flavonoids occur naturally as water-soluble glycosides. Considerable quantities of flavonoids are consumed daily in our vegetable diet, so adverse biological effects on man are not particularly intense. Indeed, there

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 171 OH HO

O

OH

R

OH

HO O2 2-oxoglutarate OH

R = H, naringenin R = OH, eriodictyol (flavanones) O2 O2 2-oxo- E1 E2 NADPH glutarate OH HO

O

R

HO O2 2-oxoglutarate

OH

E3

O

O

HO

O

O

R

HO O2 2-oxoglutarate

flavone synthase I flavone synthase II flavanone 3-hydroxylase flavonol synthase

O

R OH

– 2 H2O OH NADPH

HO

O

E7

OH

R OH

OH

R = H, (−)-epiafzelechin R = OH, (−)-epicatechin (2,3-cis-flavan-3-ols; catechins) E5: E6: E7: E8:

OH

OH HO

R = H, (+)-afzelechin R = OH, (+)-catechin (2,3-trans-flavan-3-ols; catechins)

R OH

E6 OH

E8

OH

HO O

R = H, leucopelargonidin R = OH, leucocyanidin (flavandiols; leucoanthocyanidins)

NADPH

R

OH

OH

OH

E1: E2: E3: E4:

R = H, kaempferol R = OH, quercetin (flavonols)

NADPH

O

OH

OH HO

O

OH

R

R = H, apigenin R = OH, luteolin (flavones)

R OH

OH

R = H, dihydrokaempferol R = OH, dihydroquercetin (taxifolin) (dihydroflavonols) E5

O

E4

O

OH OH

OH

R = H, pelargonidin R = OH, cyanidin (anthocyanidins)

dihydroflavanol 4-reductase anthocyanidin synthase (leucoanthocyanidin dioxygenase) anthocyanidin reductase leucoanthocyanidin reductase

Figure 4.43 is growing belief that some flavonoids are particularly beneficial, acting as antioxidants and giving protection against cardiovascular disease, certain forms of cancer, and, it is claimed, age-related degeneration of cell components. Their polyphenolic nature enables them to scavenge injurious radicals such as superoxide and hydroxyl radicals. Quercetin in particular is almost always present in substantial amounts in plant tissues; it is a powerful antioxidant, chelating metals, scavenging radicals, and preventing oxidation of low density lipoprotein. Flavonoids in red wine (quercetin, kaempferol, and anthocyanidins) and in tea (catechins and catechin gallate esters) are also demonstrated to be effective antioxidants. Green tea contains epigallocatechin gallate (Figure 4.44), a particularly effective agent. Flavonoids contribute to plant

colours: yellows from chalcones and flavonols, and reds, blues, and violets from anthocyanidins. Even the colourless materials, e.g. flavones, absorb strongly in the UV and are detectable by insects, probably aiding flower pollination. Catechins form small polymers (oligomers), the condensed tannins, e.g. the epicatechin trimer (Figure 4.44), which contribute astringency to our foods and drinks, as do the simpler gallotannins (see page 138), and are commercially important for tanning leather. Theaflavins, antioxidants found in fermented tea (see page 415), are dimeric catechin structures in which oxidative processes have led to formation of a seven-membered tropolone ring. The flavonol glycoside rutin (Figure 4.45) from buckwheat (Fagopyrum esculentum; Polygonaceae) and rue

172 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH OH

HO

O

OH

OH HO

O

OH HO

OH

O

OH

O

OH OH HO

OH

HO

O

OH

OH

OH

O

OH

O

OH

OH

OH

HO

OH

epigallocatechin gallate

OH OH

OH

O OH

O

HO OH

OH

theaflavin

OH epicatechin trimer

Figure 4.44 OH

HO HO O L-Rha

OH

OMe

O O

HO HO

O

O

OH

HO

OH

O

OH

D-Glc

O−Glc−Rha OH O quercetin

OH O hesperetin

rutinose = rhamnosyl(α1→6)glucose

rutinose

hesperidin

rutin OMe

OH O

HO D-Glc HO

O

O

O

OH

OH Rha−Glc−O

O

O HO

OH

O

HO

L-Rha OH neohesperidose = rhamnosyl(α1→2)glucose

OH hesperetin

neohesperidin

neohesperidose

O

naringenin

naringin

Figure 4.45 (Ruta graveolens; Rutaceae), and the flavanone glycoside hesperidin from Citrus peels have been included in dietary supplements as vitamin P and claimed to be of benefit in treating conditions characterized by capillary bleeding; their therapeutic efficacy is far from conclusive. Useful anti-inflammatory properties have been

demonstrated, however. Neohesperidin (Figure 4.45) from bitter orange (Citrus aurantium; Rutaceae) and naringin from grapefruit peel (Citrus paradisi) are intensely bitter flavanone glycosides. It has been found that conversion of these compounds into dihydrochalcones by hydrogenation in alkaline solution (Figure 4.46) produces

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 173 OMe Rha−Glc−O

O

Rha−Glc−O

OH

HO

H OH

O

neohesperidin

OMe OH

OH

−

OMe Rha−Glc−O

OH

OH

H2/catalyst OH

OH

O

OH

neohesperidin-chalcone

O

neohesperidin-dihydrochalcone

Figure 4.46

HO

O

OH

one-electron oxidation to radical

OH

– H+

O

–e

OH OH

HO

OH O

– H+

OH

O

OH

–e OMe

O

OMe

O

taxifolin

OH

O

OH coniferyl alcohol

radical coupling

O HO

OH

OMe

O

OH

one-electron oxidation

OH O

OH

OH OH

HO

O silybin A

OH

O

O OH

OH

OH OH

O HO

O

OH OMe

O

OMe O

H

nucleophilic attack of OH onto quinonemethide (alternative stereochemistries possible)

OH

O

silybin B (diastereoisomeric pair = silybin)

Figure 4.47 a remarkable change to their taste, and the products are now intensely sweet, being some 300–1000 times as sweet as sucrose. Neohesperidin-dihydrochalcone is used as a non-sugar sweetening agent. Flavonolignans An interesting combination of flavonoid and lignan-like structures is found in a group of compounds called flavonolignans. They arise by oxidative coupling processes between a flavonoid and a phenylpropanoid, the latter usually coniferyl alcohol. Thus, the dihydroflavonol taxifolin (dihydroquercetin) through one-electron oxidation may provide a radical, which may combine with the

radical generated from coniferyl alcohol (Figure 4.47). This would lead to an adduct which could cyclize by attack of the phenol nucleophile onto the quinone methide system provided by coniferyl alcohol. The product would be silybin, found in Silybum marianum (Compositae/Asteraceae) as a mixture of two trans diastereoisomers, silybins A and B, reflecting a lack of stereospecificity for the original radical coupling. In addition, the regioisomer isosilybin (Figure 4.48), again a mixture of trans diastereoisomers, is also found in Silybum. In keeping with the postulated biosynthetic origin, mixtures of silybin and isosilybin may be obtained by incubating taxifolin and coniferyl alcohol with a peroxidase preparation

174 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH

O

O HO

O

O

OMe

OH O

OH

OH O

OMe HO

OH

O

O OH

O

HO

OH OH

isosilybin A

O

OH

O OMe

O HO

O HO

O OH

OH

OH

OH

isosilybin B

OH OH

OH

OMe

O silychristin

(diastereoisomeric pair = isosilybin)

Figure 4.48 from Silybum marianum cells in the presence of hydrogen peroxide. Silychristin (Figure 4.48) demonstrates a further structural variant which can be seen to originate from a resonance structure of the taxifolin-derived radical, in which the unpaired electron is localized on the carbon ortho to the original 4-hydroxyl function. The flavonolignans from Silybum (milk thistle) have valuable antihepatotoxic properties and can provide protection against

liver-damaging agents [Box 4.7]. Coumarinolignans are products arising by a similar oxidative coupling mechanism which combines a coumarin with a cinnamyl alcohol and may be found in other plants. Stilbenolignans are also known; these combine a stilbene such as resveratrol (page 169) with a cinnamyl alcohol. The benzodioxane ring as seen in silybin and isosilybin is a characteristic feature of many such compounds.

Box 4.7 Silybum marianum Silybum marianum (Compositae/Asteraceae) is a biennial thistle-like plant (milk thistle) common in the Mediterranean area of Europe. The seeds yield 1.5–3% of flavonolignans collectively termed silymarin. This mixture contains mainly silybin (Figure 4.47), together with silychristin (Figure 4.48), isosilybin (Figure 4.48), and related compounds. Both silybin and isosilybin are equimolar mixtures of two trans diastereoisomers. Commercial extracts typically contain about 80% flavonolignans. Silybum marianum is widely used in traditional European medicine, the fruits being used to treat a variety of hepatic and other disorders. Silymarin has been shown to protect animal livers against the damaging effects of carbon tetrachloride, thioacetamide, drugs such as paracetamol, and the toxins α-amanitin and phalloidin found in the death cap fungus (Amanita phalloides) (see page 434). Silymarin may be used in many cases of liver disease and injury, including hepatitis, cirrhosis, and jaundice, though it still remains peripheral to mainstream medicine. It can offer particular benefit in the treatment of poisoning by the death cap fungus. These agents appear to have two main modes of action. They act on the cellular membrane of hepatocytes, inhibiting absorption of toxins; second, because of their phenolic nature, they can act as antioxidants and scavengers for radicals. Such radicals originate from liver detoxification of foreign chemicals and can cause liver damage. Derivatives of silybin with improved water solubility and/or bioavailability have been developed, e.g. the bis-hemisuccinate and a phosphatidylcholine complex.

Isoflavonoids The isoflavonoids form a quite distinct subclass of flavonoid compound, being structural variants in which

the shikimate-derived aromatic ring has migrated to the adjacent carbon of the heterocycle. This rearrangement process is brought about by a cytochrome P-450-

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 175 oxidation to radical

OH HO

O2 NADPH

O H

1,2-aryl migration

OH

HO

OH

HO

O

O Fe Enz

E1

O H R O Fe Enz R = H, liquiritigenin R = OH, naringenin (flavonoids)

R

HO

R

O

O

7

– H2O

O

HO

O

OH

OH

E3 R E1: 2-hydroxyisoflavanone synthase E2: 2,7,4′-trihydroxyisoflavanone 4′-O-methyltransferase E3, E4: 2-hydroxyisoflavanone dehydratase

4′

O

R

OH

R = H, daidzein R = OH, genistein (isoflavonoids) HO

O

O E2

– H2O

HO

OH SAM

O

OH

E4 R

O

OMe

R

O

OMe

R = H, formononetin R = OH, biochanin A (isoflavonoids)

Figure 4.49 dependent enzyme requiring NADPH and O2 cofactors, which transforms the flavanones liquiritigenin or naringenin into the isoflavones daidzein or genistein respectively via intermediate hydroxyisoflavanones (Figure 4.49). A radical mechanism has been proposed. Formation of isoflavones formononetin and biochanin A involves methylation at the hydroxyisoflavanone stage; the dehydratase enzymes appear to be specific for either hydroxy or methoxy substrates. The rearrangement step is quite rare in nature, and although flavonoids are found throughout the plant kingdom, isoflavonoids are almost entirely restricted to the Leguminosae/Fabaceae plant family. Nevertheless, many hundreds of different isoflavonoids have been identified, and structural complexity is brought about by hydroxylation and alkylation reactions, varying the oxidation level of the heterocyclic ring, or forming additional heterocyclic rings. Some of the many variants are shown in Figure 4.50; though no details are shown, the transformations, enzymes involved, and most of the genes have been characterized. Pterocarpans, e.g. medicarpin from lucerne (Medicago sativa) and pisatin from pea (Pisum sativum), and isoflavans, e.g. vestitol from lucerne, have antifungal activity and form part of the natural defence mechanism against fungal attack in these plants. Simple isoflavones (such as daidzein) and coumestans (such

as coumestrol) from lucerne and clovers (Trifolium species) have sufficient oestrogenic activity to affect the reproduction of grazing animals seriously and are termed phyto-oestrogens [Box 4.8]. These planar molecules undoubtedly mimic the shape and polarity of the steroid hormone estradiol (see page 291). The consumption of legume fodder crops by animals must, therefore, be restricted, or low isoflavonoid-producing strains have to be selected. Isoflavonoids in the human diet, e.g. from soya (Glycine max) products, are believed to give some protection against oestrogen-dependent cancers such as breast cancer, by restricting the availability of the natural hormone. In addition, they are employed as dietary oestrogen supplements for the reduction of menopausal symptoms, in a similar way to hormone replacement therapy (HRT; see page 294). The rotenoids take their name from the first known example rotenone, and are formed by ring cyclization of a methoxyisoflavone (Figure 4.51). Rotenone itself contains a C5 isoprene unit (as do virtually all the natural rotenoids) introduced via dimethylallylation of demethylmunduserone. The isopropenylfurano system of rotenone and the dimethylpyrano of deguelin are formed via rotenonic acid (Figure 4.51) without any detectable epoxide or hydroxy intermediates (compare furocoumarins,

176 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition HO

O

MeO

OH

O OH O H

O

HO

OH

O

HO

O

O

pisatin (pterocarpan) HO

O

O H

O

H

O

OH

daidzein (isoflavone)

OMe

formononetin (isoflavone)

O

OMe

medicarpin (pterocarpan)

H HO

O

O

O

O

O

O

OH

coumestrol (coumestan)

H

HO

O

O H HO

OMe

rotenone (rotenoid)

OMe

OMe

vestitol (isoflavan)

Figure 4.50

HO

O

OMe

O

O

O

O

H O

OMe OMe

OMe

hydride addition (reduction)

C-alkylation at activated position ortho to phenol

cyclization to 5-membered ring

OPP OMe HO

OMe

O

H

DMAPP

HO

O

O

O

H

cyclization to 6-membered ring

H

H O

O

E1

H

deguelin

O

O H

OMe

H

rotenone

Figure 4.51

O

H O

HO

O

OMe

O

O

O

OMe

H O

oxidation of OMe group (hydroxylation and loss of hydroxide, compare Figure 2.22) HO

O

H OMe

OMe rotenonic acid

demethylmunduserone

OMe OMe

OMe

OMe

E1: deguelin cyclase

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 177 provides an excellent biodegradable insecticide and is used as such either in pure or powdered plant form. Roots of Derris elliptica or Lonchocarpus species are rich sources of rotenone [Box 4.8].

page 164). Rotenone and other rotenoids are powerful insecticidal and piscicidal (fish poison) agents, interfering with oxidative phosphorylation. They are relatively harmless to mammals unless they enter the bloodstream, being metabolized rapidly upon ingestion. Rotenone thus

Box 4.8 Phyto-oestrogens Phyto-oestrogen (phytoestrogen) is a term applied to non-steroidal plant materials displaying oestrogenic properties. Pre-eminent amongst these are isoflavonoids. These planar molecules mimic the shape and polarity of the steroid hormone estradiol (Figure 4.52; see page 290), and are able to bind to an oestrogen receptor, though their activity is much less than that of estradiol. In some tissues, they stimulate an oestrogenic response, whilst in others they can antagonize the effect of oestrogens. Such materials taken as part of the diet, therefore, influence overall oestrogenic activity in the body by adding their effects to normal levels of steroidal oestrogens (see page 293). Foods rich in isoflavonoids are valuable in countering some of the side-effects of the menopause in women, such as hot flushes, tiredness, and mood swings. In addition, there is mounting evidence that phyto-oestrogens also provide a range of other beneficial effects, helping to prevent heart attacks and other cardiovascular diseases, protecting against osteoporosis, lessening the risk of breast and uterine cancer, and in addition displaying significant antioxidant activity which may reduce the risk of Alzheimer’s disease. Whilst some of these benefits may be obtained by the use of steroidal oestrogens, particularly via HRT (see page 294), phyto-oestrogens offer a dietary alternative. The main food source of isoflavonoids is the soya bean (Glycine max; Leguminosae/Fabaceae) (see also page 255), which contains significant levels of the isoflavones daidzein and genistein (Figure 4.52), in free form and as their 7-O-glucosides. Total isoflavone levels fall in the range 0.1–0.4%, according to variety. Soya products such as soya milk, soya flour, tofu, and soya-based textured vegetable protein may all be used in the diet for their isoflavonoid content. Breads in which wheat flour is replaced by soya flour are also popular. Extracts from red clover (Trifolium pratense; Leguminosae/Fabaceae) are also used as a dietary supplement. Red clover isoflavones are predominantly formononetin (Figure 4.50) and daidzein, together with their 7-O-glucosides. The lignans enterodiol and enterolactone (Figure 4.52) are also regarded as phyto-oestrogens. These compounds are produced by the action of intestinal microflora on lignans such as secoisolariciresinol or matairesinol ingested in the diet (see Figure 4.21, page 154). A particularly important precursor is secoisolariciresinol diglucoside from flaxseed (Linum usitatissimum; Linaceae), and flaxseed may be incorporated into foodstuffs along with soya products. Enterolactone and enterodiol were first detected in human urine, and their origins were traced back to dietary fibre-rich foods. Levels in the urine were much higher in vegetarians; a lower incidence of breast cancer in vegetarians has been related to these compounds. Enterolactone has been detected in sewage treatment plant water and even in tap water; some may arise from degradation of plant material and some from human excretions. Several other biological activities are reported for isoflavones. One of significance is the modest anticancer activity of the isoflavone genistein. This has led to development of the simple isoflavene phenoxodiol (Figure 4.52), which has a promising profile in clinical trials. Phenoxodiol targets and inhibits NADH oxidase (tNOX) in cancer cells, which in turn leads to apoptosis; it is being investigated against late-stage chemoresistant ovarian cancers, as well as prostate and cervical cancers.

HO HO

O

7

O

HO H

HO

O

H O

R

O

H OH OH

R = H, daidzein R = OH, genistein

Figure 4.52

estradiol

OH enterolactone

OH phenoxodiol

178 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 4.8 (continued) Derris and Lonchocarpus Species of Derris (e.g. Derris elliptica, Derris malaccensis) and Lonchocarpus (e.g. Lonchocarpus utilis, Lonchocarpus urucu) (Leguminosae/Fabaceae) have provided useful insecticides for many years. Roots of these plants have been employed as a dusting powder, or extracts have been formulated for sprays. Derris plants are small shrubs cultivated in Malaysia and Indonesia, whilst Lonchocarpus includes shrubs and trees, with commercial material coming from Peru and Brazil. The insecticidal principles are usually supplied as a black, resinous extract. Both Derris and Lonchocarpus roots contain 3–10% of rotenone (Figure 4.51) and smaller amounts of other rotenoids, e.g. deguelin (Figure 4.51). The resin may contain rotenone (about 45%) and deguelin (about 20%). Rotenone and other rotenoids interfere with oxidative phosphorylation, blocking transfer of electrons to ubiquinone (see page 178) by complexing with NADH:ubiquinone oxidoreductase of the respiratory electron transport chain. However, unless they enter the bloodstream they are relatively innocuous to mammals, being metabolized rapidly upon ingestion. Insects and also fish seem to lack this rapid detoxification. The fish poison effect has been exploited for centuries in a number of tropical countries, allowing lazy fishing by the scattering of powdered plant material on the water. The dead fish were collected and, when subsequently eaten, produced no ill effects on the consumers. More recently, rotenoids have been used in fish management programmes to eradicate undesirable fish species prior to restocking with other species. As insecticides, the rotenoids still find modest use, and are valuable for their selectivity and rapid biodegradability. However, they are perhaps inactivated too rapidly in the presence of light and air to compete effectively with other insecticides, such as the modern pyrethrin derivatives (see page 205).

TERPENOID QUINONES Quinones are potentially derivable by oxidation of suitable phenolic compounds, catechols (1,2dihydroxybenzenes) giving rise to ortho-quinones and quinols (1,4-dihydroxybenzenes) yielding para-quinones (see page 26). Accordingly, quinones can be formed from phenolic systems generated by either the acetate or shikimate pathways, provided a catechol or quinol system has been elaborated, and many examples are found in nature. A range of quinone derivatives and related structures containing a terpenoid fragment, as well as a shikimate-derived portion, are also widely distributed. Many of these have important biochemical functions in electron transport systems for respiration or photosynthesis, and some examples are shown in Figure 4.53. Ubiquinones (coenzyme Q; Figure 4.53) are found in almost all organisms and function as electron carriers for the electron transport chain in mitochondria. The length of the terpenoid chain is variable (n = 1–12), and species dependent, but most organisms synthesize a range of compounds, of which those where n = 7–10 usually predominate. The human redox carrier is coenzyme Q10 . They are derived from 4-hydroxybenzoic acid (Figure 4.54), though the origin of this compound varies according to the organism (see pages 140 and 157). Thus, bacteria are known to transform chorismic acid by enzymic elimination of pyruvic acid, whereas plants and animals utilize a route from phenylalanine or tyrosine via 4-hydroxycinnamic acid. 4-Hydroxybenzoic acid is the

substrate for C-alkylation ortho to the phenol group with a polyisoprenyl diphosphate of appropriate chain length (see page 306). The product then undergoes further elaboration, the exact sequence of modifications, i.e. hydroxylation, O-methylation, and decarboxylation, varying in eukaryotes and prokaryotes. Quinone formation follows in an O2 -dependent combined hydroxylation–oxidation process, and ubiquinone production then involves further hydroxylation, plus O- and C-methylation reactions. In yeast, Saccharomyces cerevisiae, nine genes have been identified as necessary for coenzyme Q biosynthesis, though not all of the encoded proteins have enzymatic functions. Coenzyme Q supplementation has been shown to be effective in treating patients with specific respiratory chain defects and to slow the progression of Parkinson’s disease symptoms. Plastoquinones (Figure 4.53) bear considerable structural similarity to ubiquinones, but are not derived from 4-hydroxybenzoic acid. Instead, they are produced from homogentisic acid, a phenylacetic acid derivative formed from 4-hydroxyphenylpyruvic acid by a very complex reaction involving decarboxylation, O2 -dependent hydroxylation, and subsequent migration of the –CH2 CO2 H side-chain to the adjacent position on the aromatic ring, all catalysed by a single enzyme (Figure 4.55). C-Alkylation of homogentisic acid ortho to a phenol group follows, and involves a polyisoprenyl diphosphate with n = 3–10, but most commonly with n = 9, i.e. solanesyl diphosphate. However, during the alkylation reaction, the –CH2 CO2 H side-chain of homogentisic acid suffers decarboxylation

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 179

O

O

MeO

R2

n = 1–12

MeO n

O

n = 3–10

H

n

O

ubiquinone-n (coenzyme Qn)

HO R1

H

O

H 3

plastoquinone-n R1 = R2 = Me, α-tocopherol R1 = H, R2 = Me, β-tocopherol R1 = Me, R2 = H, γ-tocopherol R1 = R2 = H, δ-tocopherol

O

O

n = 1–13

(vitamin E)

H 3

O

n

O menaquinone-n (vitamin K2)

phylloquinone (vitamin K1; phytomenadione)

H

Figure 4.53 CO2H DMAPP + IPP

H plants/ animals OH 4-coumaric acid

CO2H

CO2H

E3 C-alkylation with a polyisoprenyl PP

bacteria

CO2H

E1 CH3COCO2H O

E2

OPP n

OH 4-hydroxybenzoic acid

nH

OH

in eukaryotes: 1. hydroxylation 2. O-methylation E4 3. decarboxylation

CO2H

in prokaryotes: 1. decarboxylation E5 2. hydroxylation E6 3. O-methylation E7

O2 SAM

OH chorismic acid MeO

nH

OH 1. C-methylation 2. hydroxylation 3. O-methylation

O MeO MeO O ubiquinone-n E1: E2: E3: E4: E5:

nH

SAM

O2

SAM

E4 E7

E10

E9

chorismate lyase (UbiC) polyprenyl diphosphate synthase (Coq1, IspB) 4-hydroxybenzoate polyprenyltransferase (Coq2, UbiA) Coq3 UbiD

Figure 4.54

E8 O

MeO O

E6: UbiB E7: UbiG E8: Coq6, UbiH E9: Coq5, UbiE E10: Coq7, UbiF

O2

oxidation to quinone

n

H

Coq: Saccharomyces cerevisiae (n = 6) Ubi, Isp: Escherichia coli (n = 8)

180 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

HO

a complex sequence involving hydroxylation, migration of side-chain, and decarboxylation O HO O2 E1

C-alkylation ortho to phenol; also decarboxylation

H n n = 9, solanesyl PP

PPO

H n

HO

OH O

CO2H

OH

E2

CO2

4-hydroxyphenylpyruvic acid

C-methylation ortho to phenol

CO2

HO2C homogentisic acid PPO

E4

3 phytyl PP

CO2

H

HO

n

HO

OH H

O H cyclization to 6-membered ring via protonation of double bond E5

SAM

C-methylation ortho to phenol H

O

C-methylation ortho to phenol

HO

H

O H

δ-tocopherol E5

cyclization to 6-membered ring via protonation of double bond

β-tocopherol

SAM HO

H 3

plastoquinone-n

3

HO O

H

O

H 3

SAM

oxidation of quinol to quinone

n HO

E6

O

3 E3

HO O

SAM

E3 H

H

O

3

γ-tocopherol

E1: 4-hydroxyphenylpyruvate dioxygenase E2: homogentisate solanesyltransferase E3: 2-methyl-6-phytyl-1,4-benzoquinol methyltransferase

E6

C-methylation ortho to phenol

O

H 3

α-tocopherol

E4: homogentisate phytyltransferase E5: tocopherol cyclase E6: γ-tocopherol methyltransferase

Figure 4.55 and the product is thus an alkyl methyl p-quinol derivative. Further aromatic methylation (via SAM) and oxidation of the p-quinol to a quinone follow to yield the plastoquinone. Thus, only one of the two methyl groups on the quinone ring of the plastoquinone is derived from SAM. Plastoquinones are involved in the photosynthetic electron transport chain in plants. Tocopherols are also frequently found in the chloroplasts and constitute members of the vitamin E group [Box 4.9]. Their biosynthesis shares many of the features of plastoquinone biosynthesis, with an additional cyclization reaction involving the p-quinol and the terpenoid side-chain to give a chroman ring (Figure 4.55). Thus, the tocopherols, e.g. α-tocopherol and γ-tocopherol, are

not in fact quinones, but are indeed structurally related to plastoquinones. The isoprenoid side-chain added, from phytyl diphosphate, contains only four isoprene units, and three of the expected double bonds have suffered reduction. Again, decarboxylation of homogentisic acid co-occurs with the alkylation reaction. C-Methylation steps using SAM and the cyclization of the p-quinol to γ-tocopherol have been established as in Figure 4.55. Formation of δ-tocopherol and β-tocopherol occurs via a sequence in which cyclization precedes the methylation. Note again that one of the nuclear methyl groups is homogentisate-derived, whilst the others are supplied by SAM. A number of the enzymes characterized display broad substrate specificity.

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 181

Box 4.9 Vitamin E Vitamin E refers to a group of fat-soluble vitamins, the tocopherols, e.g. α-, β-, γ-, δ-tocopherols (Figure 4.55), which are widely distributed in plants, with high levels in cereal seeds such as wheat, barley, and rye. Wheat germ oil is a particularly good source. The proportions of the individual tocopherols vary widely in different seed oils, e.g. principally β- in wheat oil, γ- in corn oil, α- in safflower oil, and γ- and δ- in soybean oil. Vitamin E deficiency is virtually unknown, with most of the dietary intake coming from food oils and margarine, though much can be lost during processing and cooking. Rats deprived of the vitamin display reproductive abnormalities. α-Tocopherol has the highest activity (100%), with the relative activities of β-, γ-, and δ-tocopherols being 50%, 10%, and 3% respectively. The acetate ester α-tocopheryl acetate is the main commercial form used for food supplementation and for medicinal purposes. The vitamin is known to provide valuable antioxidant properties, probably preventing the destruction by radical reactions of vitamin A and unsaturated fatty acids in biological membranes. It is used commercially to retard rancidity in fatty materials in food manufacturing, and there are also claims that it can reduce the effects of ageing and help to prevent heart disease. Its antioxidant effect is likely to arise by reaction with peroxyl radicals, generating by one-electron phenolic oxidation a resonance-stabilized radical that does not propagate the radical reaction. Instead, it mops up further peroxyl radicals (Figure 4.56). In due course, the tocopheryl peroxide is hydrolysed to the tocopherolquinone. A main function of vitamin C (see page 492) is to provide a regenerating system for tocopherol, allowing the tocopheryloxyl radical to abstract a hydrogen atom.

initiation of radical reaction by peroxy radical HO

ROO

O

O

O

O

α-tocopherol

quenching of second peroxy radical O ROO

resonance-stabilized radical

O OOR

O

α-tocopheryloxyl radical OH

loss of peroxide leaving group

hydrolysis of hemiketal

O

O O α-tocopherolquinone

H 2O O OH

O O

formation of hemiketal

OH2

Figure 4.56

The phylloquinones (vitamin K1 ) and menaquinones (vitamin K2 ) are shikimate-derived naphthoquinone derivatives found in plants and algae (vitamin K1 ) or bacteria and fungi (vitamin K2 ) [Box 4.10]. The most common phylloquinone structure (Figure 4.53) has a diterpenoid (C20 ) side-chain, whereas the range of menaquinone structures tends to be rather wider, with 1–13 isoprene units. These quinones are derived from chorismic acid via its isomer isochorismic acid (Figure 4.57). Additional carbon atoms for the naphthoquinone skeleton are provided by 2-oxoglutaric acid, which is incorporated by a mechanism involving the coenzyme thiamine diphosphate (TPP; see page 24).

2-Oxoglutaric acid is decarboxylated in the presence of TPP to give the TPP anion of succinic semialdehyde, which attacks isochorismic acid in a Michael-type reaction. Loss of the thiamine cofactor, elimination of pyruvic acid, and then dehydration yields the intermediate o-succinylbenzoic acid (OSB); the dehydration is an unusual syn-elimination, and is suggested to involve initial proton removal with carboxylate stabilization of the anion. OSB is activated by formation of a coenzyme A ester, and a Dieckmann-like condensation allows ring formation. The dihydroxynaphthoic acid coenzyme A ester is the more favoured aromatic tautomer, and hydrolysis of this leads to the free acid. This compound

182 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

HO2C

HO2C

OH2 O

CO2H

H HO chorismic acid

Michael-type addition

O

OH

HO2C

OH O

O

CO2H

OH CO2H

E2

E1 HO

isochorismic acid

TPP-dependent decarboxylation of a-keto acid to aldehyde (see Figure 2.16); nucleophilic addition of TPP anion onto HO2C aldehyde then allows removal of aldehydic proton which has become acidic

TPP TPP

− CO2

CO2H succinic semi-aldehyde −TPP anion

H O TPP H HO2C

CO2H

E2 OH CO2H

O

O 2-oxoglutaric acid

H

B suggested mechanism for syn-elimination OH H O

H

OH

O 2-succinyl-5-enolpyruvyl-6-hydroxy3-cyclohexene-1-carboxylic acid (SEPHCHC)

O OH

OH

E3

O

Claisen-like condensation (Dieckmann reaction) O COSCoA

C

COSCoA HSCoA ATP

OSB-CoA OH

OH

CO2H CO H 2

CO2H

CO2H E4 syn-dehydration to O O form aromatic ring o-succinyl 2-succinyl-6-hydroxy-2,4benzoic acid cyclohexadiene-1-carboxylic acid (OSB) (SHCHC)

E5 O

enolization

1,4-elimination of pyruvic acid

OH

OH

E6 O

CO2H

CO2H

COSCoA

H OH

E7 hydrolysis of thioester

PPO OH 1,4-dihydroxy2-naphthoic acid

n polyisoprenyl PP

E8

O

C-alkylation with concomitant decarboxylation (see Figure 4.58)

E1: isochorismate synthase (menF) E2: 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene1-carboxylate synthase (men D) E3: 2-succinyl-6-hydroxy-2,4-cyclohexadiene1-carboxylate synthase (menH) E4: o-succinylbenzoate synthase (men C)

Figure 4.57

O H n

CO2

H

SAM n

E9

C-methylation O

E5: E6: E7: E8: E9:

O menaquinone-n (vitamin K2) o-succinylbenzoyl-CoA synthetase (men E) 1,4-dihydroxy-2-naphthoyl-CoA synthase (menB) thioesterase 1,4-dihydroxy-2-naphthoate phytyltransferase (menA) demethylmenaquinone methyltransferase (menG, ubiE)

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 183

R

PPO R

OH

O

H

decarboxylation of β-keto acid

O

CO2H

oxidation to quinone

OH

O

R O

O R

OH

alkylation ortho to phenol

CO2

OH

O

OH

Figure 4.58 is now the substrate for alkylation and methylation, as seen with ubiquinones and plastoquinones. However, the terpenoid fragment is found to replace the carboxyl group, and the decarboxylated analogue is not involved. The transformation of 1,4-dihydroxynaphthoic acid into the isoprenylated naphthoquinone appears to be catalysed by a single enzyme and can be rationalized

by the mechanism in Figure 4.58. This involves alkylation, decarboxylation of the resultant β-keto acid, and finally an oxidation to the p-quinone. OSB and 1,4-dihydroxynaphthoic acid have been implicated in the biosynthesis of a wide range of plant naphthoquinones and anthraquinones; other carbon atoms in the skeleton derive from a C5 isoprene unit.

Box 4.10 Vitamin K OH

O

O

O

O2 E2

E1

OH O O

OH vitamin KH2 (quinol)

O vitamin K

HO

O O

E1 O

O − H2O

O

O

O O

HO

O vitamin K epoxide

O

OH

HO

O

HO

O

O

(B−),

strong base ionizes glutamate residue to enolate anion OP

HN

HN

HN

O

O

O CO2

HO2C HO2C

NH

−γ-carboxyglutamate−

Figure 4.59

HO2C

NH

HO2C

H

E2

NH

H O C O

B

−glutamate− (prothrombin)

OP menadiol phosphate E1: vitamin K epoxide reductase E2: vitamin K-dependent carboxylase

184 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 4.10 (continued) Vitamin K comprises a number of lipid-soluble naphthoquinone derivatives, with vitamin K1 (phylloquinone; Figure 4.53) being of plant origin whilst the vitamins K2 (menaquinones) are produced by microorganisms. Dietary vitamin K1 is obtained from almost any green vegetable, whilst a significant amount of vitamin K2 is produced by the intestinal microflora. As a result, vitamin K deficiency is rare and only necessary where prolonged use of antibiotics has destroyed intestinal bacteria, or where absorption of nutrients from the intestine is impaired. Vitamin K1 (phytomenadione) or the water-soluble menadiol phosphate (Figure 4.59) may be employed as supplements. Menadiol is oxidized in the body to the quinone, which is then alkylated, e.g. with geranylgeranyl diphosphate, to yield a metabolically active product, and would be employed where malabsorption is a problem. Vitamin K supplements are given routinely to newborn infants, since they initially lack the intestinal bacteria capable of producing the vitamin. Vitamin K is involved in normal blood clotting processes, and a deficiency would lead to haemorrhage. Blood clotting requires the carboxylation of glutamate residues in the protein prothrombin, generating bidentate ligands that allow the protein to bind to other factors. This carboxylation requires carbon dioxide, molecular oxygen, and the reduced quinol form of vitamin K. A mechanism for the process based on current information is shown in Figure 4.59. During the carboxylation, the reduced vitamin K is converted into an epoxide, and vitamin K is subsequently regenerated by reduction. The same protein is capable of reducing vitamin K to its quinol form. Anticoagulants such as dicoumarol and warfarin (see page 163) inhibit this reductase. In contrast, the polysaccharide anticoagulant heparin (see page 498) does not interfere with vitamin K metabolism, but acts by complexing with blood-clotting enzymes.

Folic acid FURTHER READING Shikimate Pathway Alibhai MF and William C. Stallings WC (2001) Closing down on glyphosate inhibition – with a new structure for drug discovery. Proc Natl Acad Sci USA 98, 2944–2946. Anderson KS (2005) Detection of novel enzyme intermediates in PEP-utilizing enzymes. Arch Biochem Biophys 433, 47–58. Knaggs AR (2003) The biosynthesis of shikimate metabolites. Nat Prod Rep 20, 119–136. Mustafa NR and Verpoorte R (2005) Chorismate derived C6 C1 compounds in plants. Planta 222, 1–5.

DellaPenna D (2007) Biofortification of plant-based food: enhancing folate levels by metabolic engineering. Proc Natl Acad Sci USA 104, 3675–3676. Finglas PM and Wright AJA (2002) Folate bioavailability and health. Phytochem Rev 1, 189–198. Hanson AD and Gregory JF (2002) Synthesis and turnover of folates in plants. Curr Opin Plant Biol 5, 244–249. Kompis IM, Islam K and Then RL (2005) DNA and RNA synthesis: antifolates. Chem Rev 105, 593–620.

Melanins Roy S (2007) Melanin, melanogenesis, and vitiligo. Prog Chem Org Nat Prod 88, 131–185.

Chloramphenicol Tamiflu Farina V and Brown JD (2006) Tamiflu: the supply problem. Angew Chem Int Ed 45, 7330–7334.

Tannins Feldman KS (2005) Recent progress in ellagitannin chemistry. Phytochemistry 66, 1984–2000. Haslam E (2007) Vegetable tannins – lessons of a phytochemical lifetime. Phytochemistry 68, 2713–2721. Khanbabaee K and van Ree T (2001) Tannins: classification and definition. Nat Prod Rep 18, 641–649. Niemetz R and Gross GG (2005) Enzymology of gallotannin and ellagitannin biosynthesis. Phytochemistry 66, 2001–2011. Okuda T (2005) Systematics and health effects of chemically distinct tannins in medicinal plants. Phytochemistry 66, 2012–2031.

He J, Magarvey N, Piraee M and Vining LC (2001) The gene cluster for chloramphenicol biosynthesis in Streptomyces venezuelae ISP5230 includes novel shikimate pathway homologues and a monomodular non-ribosomal peptide synthetase gene. Microbiology 147, 2817–2829. Pacholec M, Sello JK, Walsh CT and Thomas MG (2007) Formation of an aminoacyl-S-enzyme intermediate is a key step in the biosynthesis of chloramphenicol. Org Biomol Chem 5, 1692–1694.

Phenylpropanoids Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi AB, Rouabhia M, Mahdouani K and Bakhrouf A (2007) The chemical composition and biological activity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L. Myrtaceae): a short review. Phytother Res 21, 501–506.

The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids 185 Dixon RA, Chen F, Guo D and Parvathi K (2001) The biosynthesis of monolignols: a ‘metabolic grid’, or independent pathways to guaiacyl and syringyl units? Phytochemistry 57, 1069–1084. Dixon RA and Reddy MSS (2003) Biosynthesis of monolignols. Genomic and reverse genetic approaches. Phytochem Rev 2, 289–306. Petersena M and Simmonds MSJ (2003) Molecules of interest. Rosmarinic acid. Phytochemistry 62, 121–125. Walton NJ, Mayer MJ and Narbad A (2003) Molecules of interest. Vanillin. Phytochemistry 63, 505–515.

Lignin Anterola AM and Lewis NR (2002) Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 61, 221–294. Boerjan W, Ralph J and Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54, 519–546. Hatfield R and Vermerris W (2001) Lignin formation in plants. The dilemma of linkage specificity. Plant Physiol 126, 1351–1357. Ralph J, Lundquist K, Brunow G, Lu F, Kim H, Schatz PF, Marita JM, Hatfield RD, Ralph SA, Christensen JH and Boerjan W (2004) Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem Rev 3, 29–60.

Lignans Apers S, Vlietinck SA and Pieters L (2003) Lignans and neolignans as lead compounds. Phytochem Rev 2, 201–217. Botta B, Delle Monache G, Misiti D, Vitalia A and Zappia G (2001) Aryltetralin lignans: chemistry, pharmacology and biotransformations. Curr Med Chem 8, 1363–1381. Davin LB and Lewis NG (2003) An historical perspective on lignan biosynthesis: monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism. Phytochem Rev 2, 257–288. Fuss E (2003) Lignans in plant cell and organ cultures: an overview. Phytochem Rev 2, 307–320. Harmatha J and Dinan L (2003) Biological activities of lignans and stilbenoids associated with plant-insect chemical interactions. Phytochem Rev 2, 321–330. Saleem M, Kim HJ, Ali MS and Lee YS (2005) An update on bioactive plant lignans. Nat Prod Rep 22, 696–716. Smeds AI, Willf¨or SM, Pietarinen SP, Peltonen-Sainio P and Reunanen MHT (2007) Occurrence of ‘mammalian’ lignans in plant and water sources. Planta 226, 639–646. Umezawa T (2003) Diversity in lignan biosynthesis. Phytochem Rev 2, 371–390. Westcott ND and Muir AD (2003) Flax seed lignan in disease prevention and health promotion. Phytochem Rev 2, 401–417.

Podophyllum Castro MA, del Corral JMM, Gordaliza M, G´omez-Zurita MA, Garc´ıa PA and San Feliciano A (2003) Chemoinduction of

cytotoxic selectivity in podophyllotoxin-related lignans. Phytochem Rev 2, 219–233. Lee K-H and Xiao Z (2003) Lignans in treatment of cancer and other diseases. Phytochem Rev 2, 341–362. Meresse P, Dechaux E, Monneret C and Bertounesque E (2004) Etoposide: discovery and medicinal chemistry Curr Med Chem 11, 2443–2466.

Coumarins Bourgaud F, Hehn A, Larbat R, Doerper S, Gontier E, Kellner S and Matern U (2006) Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem Rev 5, 293–308.

Kava Abe I, Watanabe T and Noguchi H (2004) Enzymatic formation of long-chain polyketide pyrones by plant type III polyketide synthases. Phytochemistry 65, 2447–2453. Singh YN (2005) Potential for interaction of kava and St. John’s wort with drugs. J Ethnopharmacol 100, 108–113. Whittona PA, Laua A, Salisbury A, Whitehouse J and Evans CS (2003) Kava lactones and the kava-kava controversy. Phytochemistry 64, 673–679.

Flavonoids Ayabe S-I and Akashi T (2006) Cytochrome P450s in flavonoid metabolism. Phytochem Rev 5, 271–282. De Pascual-Teresa S and Sanchez-Ballesta MT (2008) Anthocyanins: from plant to health. Phytochem Rev 7, 281–299. Garg, A, Garg S, Zaneveld LJD and Singla AK (2001) Chemistry and pharmacology of the citrus bioflavonoid hesperidin. Phytother Res 15, 655–669. Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57, 761–780. Hackman RM, Polagruto JA, Zhu QY, Sun B, Fujii H and Keen CL (2008) Flavanols: digestion, absorption and bioactivity. Phytochem Rev 7, 195–208. Kong J-M, Chia L-S, Goh N-K, Chia T-F and Brouillard R (2003) Analysis and biological activities of anthocyanins. Phytochemistry 64, 923–933. Martens S and Mith¨ofer A (2005) Flavones and flavone synthases. Phytochemistry 66, 2399–2407; corrigendum 67, 521. Nagle DG, Ferreira D and Zhou Y-D (2006) Epigallocatechin-3-gallate (EGCG): chemical and biomedical perspectives. Phytochemistry 67, 1849–1855. Prior RL and Gu L (2005) Occurrence and biological significance of proanthocyanidins in the American diet. Phytochemistry 66, 2264–2280. Rice-Evans C (2001) Flavonoid antioxidants. Curr Med Chem 8, 797–807. Schijlen EGWM, de Vos CHR, van Tunen AJ and Bovy AG (2004) Modification of flavonoid biosynthesis in crop plants. Phytochemistry 65, 2631–2648. Springob K, Nakajima J-I, Yamazaki M and Saito K (2003) Recent advances in the biosynthesis and accumulation of anthocyanins. Nat Prod Rep 20, 288–303.

186 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Winkel-Shirley B (2001) Flavonoid biosynthesis. a colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126, 485–493. Xie D-Y and Dixon RA (2005) Proanthocyanidin biosynthesis – still more questions than answers? Phytochemistry 66, 2127–2144.

Stilbenes Cirla A and Mann J (2003) Combretastatins: from natural products to drug discovery. Nat Prod Rep 20, 558–564. Nam N-H (2003) Combretastatin A-4 analogues as antimitotic antitumor agents. Curr Med Chem 10, 1697–1722. Roberti M, Pizzirani D, Simoni D, Rondanin R, Baruchello R, Bonora C, Buscemi F, Grimaudo S and Tolomeo M (2003) Synthesis and biological evaluation of resveratrol and analogues as apoptosis-inducing agents. J Med Chem 46, 3546–3554. Tron GC, Pirali T, Sorba G, Pagliai F, Busacca S and Genazzani AA (2006) Medicinal chemistry of combretastatin A4: present and future directions. J Med Chem 49, 3033–3044.

Flavonolignans Crocenzi FA and Roma MG (2006) Silymarin as a new hepatoprotective agent in experimental cholestasis: new possibilities for an ancient medication. Curr Med Chem 13, 1055–1074. Gaˇza´ k R, Walterov´a D and Kren V (2007) Silybin and silymarin – new and emerging applications in medicine. Curr Med Chem 14, 315–338.

Isoflavonoids, Phytoestrogens Cornwell T, Cohick W and Raskin I (2004) Dietary phytoestrogens and health. Phytochemistry 65, 995–1016. Cos P, De Bruyne T, Apers S, Van den Berghe D, Pieters L and Vlietinck AJ (2003) Phytoestrogens: recent developments. Planta Med 69, 589–599. Dixon RA (2004) Phytoestrogens. Annu Rev Plant Biol 55, 225–261. Dixon RA and Ferreira D (2002) Molecules of interest. Genistein. Phytochemistry 60, 205–211. Heinonen S-M, W¨ah¨al¨a K and Adlercreutz H (2002) Metabolism of isoflavones in human subjects. Phytochem Rev 1, 175–182.

Martin JHJ, Crotty S, Warren P and Nelson PN (2007) Does an apple a day keep the doctor away because a phytoestrogen a day keeps the virus at bay? A review of the anti-viral properties of phytoestrogens. Phytochemistry 68, 266–274. Ososki AL and Kennelly EJ (2003) Phytoestrogens: a review of the present state of research. Phytother Res 17, 845–869. Reinwald S and Weaver CM (2006) Soy isoflavones and bone health: a double-edged sword? J Nat Prod 69, 450–459. Vaya J and Tamir S (2004) The relation between the chemical structure of flavonoids and their estrogen-like activities. Curr Med Chem 11, 1333–1343. Veitch NC (2007) Isoflavonoids of the Leguminosae. Nat Prod Rep 24, 417–464.

Terpenoid Quinones Azzi A, Ricciarelli R and Zingg J-M (2002) Non-antioxidant molecular functions of α-tocopherol (vitamin E). FEBS Lett 519, 8–10. DellaPenna D (2005) A decade of progress in understanding vitamin E synthesis in plants. J Plant Physiol 162, 729–737. DellaPenna D and Pogson BJ (2006) Vitamin synthesis in plants: tocopherols and carotenoids. Annu Rev Plant Biol 57, 711–738. D¨ormann P (2007) Functional diversity of tocochromanols in plants. Planta 225, 269–276. Herbers K (2003) Vitamin production in transgenic plants. J Plant Physiol 160, 821–829. Maeda H and DellaPenna D (2007) Tocopherol functions in photosynthetic organisms. Curr Opin Plant Biol 10, 260–265. Moran GR (2005) 4-Hydroxyphenylpyruvate dioxygenase. Arch Biochem Biophys 433, 117–128. Munn´e-Bosch S and Falk J (2004) New insights into the function of tocopherols in plants. Planta 218, 323–326. Niki E and Noguchi N (2004) Dynamics of antioxidant action of vitamin E. Acc Chem Res 37, 45–51. Nohl H, Kozlov AV, Staniek K, and Gille L (2001) The multiple functions of coenzyme Q. Bioorg Chem 29, 1–13. Traber MG (2004) Vitamin E, nuclear receptors and xenobiotic metabolism. Arch Biochem Biophys 423, 6–11. Upston JM, Kritharides L and Stocker R (2003) The role of vitamin E in atherosclerosis. Prog Lipid Res 42, 405–422.

5

THE MEVALONATE AND METHYLERYTHRITOL PHOSPHATE PATHWAYS: TERPENOIDS AND STEROIDS

Terpenoids form a large and structurally diverse family of natural products derived from C5 isoprene units (Figure 5.1) joined in a head-to-tail fashion. Typical structures contain carbon skeletons represented by (C5 )n , and are classified as hemiterpenes (C5 ), monoterpenes (C10 ), sesquiterpenes (C15 ), diterpenes (C20 ), sesterterpenes (C25 ), triterpenes (C30 ), and tetraterpenes (C40 ) (Figure 5.2). Higher polymers are encountered in materials such as rubber. Isoprene itself (Figure 5.1) was known as a decomposition product from various natural cyclic hydrocarbons, and had been suggested as the fundamental building block for these compounds, also referred to as ‘isoprenoids’. Isoprene is produced naturally but is not involved in the formation of these compounds; the biochemically active isoprene units were subsequently identified as the diphosphate (pyrophosphate) esters dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) (Figure 5.2). Relatively few of the natural terpenoids conform exactly to the simple concept of a linear head-to-tail combination of isoprene units as seen with geraniol (C10 ), farnesol (C15 ), and geranylgeraniol (C20 ) (Figure 5.3). Squalene (C30 ) and phytoene (C40 ), although formed entirely of isoprene units, display a tail-to-tail linkage at the centre of the molecules. Most terpenoids are modified further by cyclization reactions, though the head-to-tail arrangement of the units can usually still be recognized, e.g. menthol,

bisabolene, and taxadiene. The linear arrangement of isoprene units can be much more difficult to appreciate in many other structures when rearrangement reactions have taken place, e.g. steroids, where, in addition, several carbon atoms have been lost. Nevertheless, such compounds are formed by way of regular terpenoid precursors. Terpenoids comprise the largest group of natural products, with over 35 000 known members. Many other natural products contain terpenoid elements in their molecules, in combination with carbon skeletons derived from other sources, such as the acetate and shikimate pathways. Many alkaloids, phenolics, and vitamins discussed in other chapters are examples of this. A particularly common terpenoid fragment in such cases is a single C5 unit, usually a dimethylallyl substituent, and molecules containing these isolated isoprene units are sometimes referred to as ‘meroterpenoids’. Some examples include furocoumarins (see page 162), rotenoids (see page 175), and ergot alkaloids (see page 387). One should also note that the term ‘prenyl’ is in general use to indicate the dimethylallyl substituent. Even macromolecules like proteins can be modified by attaching terpenoid chains. Cysteine residues in proteins are alkylated with farnesyl (C15 ) or geranylgeranyl (C20 ) groups, thereby increasing the lipophilicity of the protein and its ability to associate with membranes.

Medicinal Natural Products: A Biosynthetic Approach, 3rd Edition. Paul M. Dewick © 2009 John Wiley & Sons, Ltd. ISBN: 978-0-470-74168-9

188 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

C5 isoprene unit

isoprene

Figure 5.1 MEVALONIC ACID AND METHYLERYTHRITOL PHOSPHATE The biochemical isoprene units may be derived by two pathways: by way of intermediates mevalonic acid (MVA) (Figure 5.4) or 2-C-methyl-d-erythritol 4-phosphate (methylerythritol phosphate; MEP; see Figure 5.6). MVA, itself a product of acetate metabolism, had been established as a precursor of the animal sterol cholesterol, and the steps leading to and from MVA Mevalonic acid

OPP dimethylallyl PP (DMAPP) (C5)

were gradually detailed in a series of painstakingly executed experiments. For many years, the early parts of the mevalonate pathway were believed to be common to the whole range of natural terpenoid derivatives in all organisms. However, after detailed investigation of inconsistencies in labelling patterns, it has since been proven that an alternative pathway to IPP and DMAPP exists, via MEP, and that this pathway is probably more widely utilized in nature than is the mevalonate pathway. This pathway is also referred to as the mevalonate-independent pathway or the deoxyxylulose phosphate pathway; the terminology MEP pathway is preferred, in that MEP is the first committed terpenoid precursor, whilst deoxyxylulose phosphate is also used for the biosynthesis of pyridoxal phosphate (vitamin B6 , page 32) and thiamine (vitamin B1 , page 31). Methylerythritol phosphate

OPP isopentenyl PP (IPP) (C5)

Hemiterpenes (C5)

C10

Monoterpenes (C10) Iridoids

C15

Sesquiterpenes (C15)

C20

Diterpenes (C20)

C25

Sesterterpenes (C25)

IPP

×2

IPP

IPP ×2

C30

Triterpenoids (C30)

Steroids (C18−C30) C40

Figure 5.2

Tetraterpenes (C40) Carotenoids

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 189

OH geraniol (C10)

OH farnesol (C15)

OH geranylgeraniol (C20)

squalene (C30)

phytoene (C40)

OH

menthol (C10)

bisabolene (C15)

taxadiene (C20)

Figure 5.3 Three molecules of acetyl-coenzyme A are used to form MVA. Two molecules combine initially in a Claisen condensation to give acetoacetyl-CoA, and a third is incorporated via a stereospecific aldol addition giving the branched-chain ester 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (Figure 5.4). Two of the acetyl-CoA molecules appear to be bound to the enzyme via a thiol group. One linkage is broken during the Claisen reaction and the second is subsequently hydrolysed to form the free-acid group of HMG-CoA. Note that the mevalonate pathway does not use malonyl-CoA and it thus diverges from the acetate pathway at the very first step. In the acetate pathway, an equivalent acetoacetyl thioester (bound to the acyl carrier protein, see page 67) would be formed using the thioester of malonic acid as a more nucleophilic species. In the second step of the mevalonate pathway, it should also be noted that, on purely chemical grounds, acetoacetyl-CoA is the more acidic substrate and might be expected to act as the nucleophile rather than the third acetyl-CoA molecule.

The enzyme thus achieves what is a less favourable reaction. The conversion of HMG-CoA into (3R)-MVA involves a two-step reduction of the thioester group to a primary alcohol via the aldehyde, and provides an essentially irreversible and rate-limiting transformation. Drug-mediated inhibition of this enzyme (HMG-CoA reductase) is an important means of regulating the biosynthesis of mevalonate and ultimately of the steroid cholesterol (see statins, page 98). The six-carbon compound MVA is transformed into the five-carbon phosphorylated isoprene units in a series of reactions, beginning with phosphorylation of the primary alcohol group. Two different ATP-dependent enzymes are involved, resulting in mevalonic acid diphosphate, and decarboxylation–dehydration then follows to give IPP. Whilst a third molecule of ATP is required for this last transformation, there is no evidence for phosphorylation of the tertiary hydroxyl, though this would convert the hydroxyl into a better leaving group. Hydrolysis of ATP may assist decarboxylation, as shown in Figure 5.4. IPP

190 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

O

enzyme-bound acetyl group Claisen O H reaction

EnzSH

SCoA E1 acetyl-CoA

stereospecific aldol reaction; also involves hydrolysis of acetyl–enzyme linkage

H O

O

OH O

SEnz SCoA

acetyl-CoA

SCoA acetoacetyl-CoA

E1

O

E2

EnzSH O

E2

enzyme-bound acetyl group

6

OH

1 3

2

OH

NADPH

5

OH

4

2 × ATP sequential phosphorylation of the primary alcohol to a diphosphate H

O

OH

HO2C

O

E3

E3

mevaldic acid

H mevaldic acid hemithioacetal

SCoA

E4 E5 O HO P O ADP

O

OH

OH OPP

ATP –CO2 E6

ATP facilitates the decarboxylation–elimination; phosphorylation of the tertiary alcohol to make a better leaving group is apparently not involved E1: E2: E3: E4:

NADPH

OH

HO2C

mevalonic acid (MVA)

E3

reduction of thioester to aldehyde via hemithioacetal

reduction of aldehyde to alcohol HO2C

SCoA

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)

SEnz

acetyl-CoA

+ EnzSH

HO2C

stereospecific allylic isomerization

5

H

3 4

2

1

OPP

HR H S

isopentenyl PP (IPP)

acetoacetyl-CoA synthase 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) mevalonate kinase

OPP

E7

dimethylallyl PP (DMAPP)

E5: phosphomevalonate kinase E6: mevalonate 5-diphosphate decarboxylase E7: isopentenyl diphosphate isomerase (IPP isomerase)

Figure 5.4 is isomerized to the other isoprene unit, DMAPP, by an isomerase enzyme which incorporates a proton from water onto C-4 and stereospecifically removes the pro-R proton (HR ) from C-2. This reaction is used to provide the two compounds in the amounts required for further metabolism. Two different types of isomerase enzyme have been distinguished: a type I enzyme requiring a divalent metal ion and a type II enzyme that requires a divalent metal ion together with FMN for activity. Both enzymes appear to employ a protonation–deprotonation mechanism. The conversion of IPP into DMAPP generates a reactive electrophile and, therefore, a good alkylating agent. DMAPP possesses a good leaving group, the diphosphate, and can ionize readily to yield an allylic carbocation which is stabilized by charge delocalization (Figure 5.5). In contrast, IPP with its terminal double bond is more likely to act as a nucleophile, especially towards

OPP DMAPP carbocation formation

Note: when using this representation of the allylic cation, do not forget it contains a double bond

resonance-stabilized allylic cation

Figure 5.5

the electrophilic DMAPP. These differing reactivities are the basis of terpenoid biosynthesis, and carbocations feature strongly in mechanistic rationalizations of the pathways.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 191 nucleophilic attack of enamine onto aldehyde

CO2H O

H3C

CO2

pyruvic acid thiamine PP (TPP)

R1 E1 see Figure 2.16

H

S

OH

OH

O

OH

N

H

O

H3 C

OP OH D-glyceraldehyde 3-P

R1 E1

OP

OP O

OH N

S

1-deoxy-D-xylulose 5-P

E1

R1

R2

R2 TPP/pyruvate-derived enamine

OH

N

R2

OH N O reverse aldol reaction

OH

O

aldol reaction

OH

OP E2 O H 1-deoxyD-xylulose 5-P

H

OP HO

OH

OH

P

O

OH

OH

P

O CH2 O OH HO

2-phospho-4-(CDP)2-C-methyl-D-erythritol E5

O

N

OP

OP O

E2

OH

OH OH 2-C-methylD-erythritol 4-P

compare formation E3 of UDPglucose, Figure 2.28 O

OH O

ATP

NH2 N

O

P O CH2 O O OH OH P

O E4

CTP

OH

OH

OH

4-(CDP)-2-C-methylD-erythritol

N

O

OH

HO

CMP OPP

O OH O P

isopentenyl PP (IPP)

O P

O

OH

OH H OH 2-C-methyl-D-erythritol2,4-cyclophosphate

E6

OPP

E7

OH 4-hydroxy-3-methylbut-2-enyl diphosphate

E1: 1-deoxy-D-xylulose 5-phosphate synthase (DXP synthase) E2: 2-C-methyl-D-erythritol 4-phosphate synthase; 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) E3: 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (IspD) E4: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE)

OPP H+

H

+

E8 OPP

OPP OPP dimethylallyl PP (DMAPP)

Figure 5.6

OH

NADPH

≡

nucleophilic attack of phosphate hydroxyl on diphosphate; O formation of phosphoanhydride NH2 P O OH O N O OH O

reduction of aldehyde to alcohol; the aldehyde intermediate remains enzyme-bound

fosmidomycin OH

OP O

P

O

O

TPP anion regenerated

S

E5: E6: E7: E8:

H H major product

H minor product

2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (IspG) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH) isopentenyl diphosphate isomerase (IPP isomerase)

192 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition Glycolytic pathway intermediates pyruvic acid and glyceraldehyde 3-phosphate are used in the production of MEP; the pyruvate carboxyl is lost in this process (Figure 5.6). Thiamine diphosphate-mediated decarboxylation of pyruvate (compare page 23) produces an acetaldehyde-equivalent bound in the form of an enamine. This reacts as a nucleophile in an addition reaction with the glyceraldehyde 3-phosphate. Subsequent release from the TPP carrier generates 1-deoxy-d-xylulose 5-phosphate (deoxyxylulose phosphate), which is transformed into MEP by a rearrangement process. This has been shown to involve a reverse aldol–aldol sequence (Figure 5.6), coupled with a reduction. A single enzyme catalyses these skeletal rearrangement and reduction reactions without release of any intermediate; the product now contains the branched-chain system equivalent to the isoprene unit. Reaction of MEP with cytidine triphosphate (CTP) produces a cytidine diphospho derivative (compare uridine diphosphoglucose in glucosylation, page 31), which is then phosphorylated via ATP. The resultant 2-phosphate is then converted into a cyclic phosphoanhydride with loss of cytidine phosphate. The subsequent steps leading to IPP and DMAPP are the least understood part of the pathway. Gene methodology has shown that two enzymes are involved, the first producing 4-hydroxy-3-methylbut-2-enyl diphosphate and the second converting this into predominantly IPP, but also DMAPP. Both steps are reductive in nature, but mechanisms are yet to be elucidated. The formation of both IPP and DMAPP (ratios are typically in the region 5:1 to 4:1) is suggested to involve a delocalized allylic system (radical or anion, shown in Figure 5.6 as an anion), with protons being supplied by water. Although this pathway coproduces IPP and DMAPP, isomerism of IPP to DMAPP as in the mevalonate pathway is also possible to balance the pool sizes of these intermediates. Whether the mevalonate pathway or the MEP pathway supplies isoprene units for the biosynthesis of a particular terpenoid must be established experimentally. This can be determined from the results of feeding [1-13 C]-d-glucose as precursor; this leads to different labelling patterns in the isoprene unit according to the pathway operating (Figure 5.7). Animals and fungi appear to lack the MEP pathway, so utilize the mevalonate pathway exclusively. The MEP pathway is present in plants, algae, and most bacteria. Plants and some bacteria are equipped with and employ both pathways, often concurrently. In plants, the two pathways appear to be compartmentalized, so that the mevalonate pathway enzymes are localized in the cytosol, whereas the MEP pathway enzymes are found in chloroplasts. The cytosolic pool of IPP serves as a precursor of C15 derivatives (farnesyl PP (FPP) and sesquiterpenes; see

Figure 5.2), and in due course triterpenoids and steroids (2 × FPP). Accordingly, triterpenoids and steroids, and some sesquiterpenoids (cytosolic products) are formed by the mevalonate pathway, whilst most other terpenoids (C10 , C20 , C40 ) are formed in the chloroplasts and are MEP derived. Of course there are exceptions. There are also examples where the two pathways can supply different portions of a molecule, or where there is exchange of late-stage common intermediates between the two pathways (cross-talk), resulting in a contribution of isoprene units from each pathway. In the following part of this chapter, these complications will not be considered further, and in most cases there is no need to consider the precise source of the isoprene units. An area of special pharmacological interest where the early pathway is of particular concern is steroid biosynthesis, which appears to be from mevalonate in the vast majority of organisms. Thus, inhibitors of mevalonate pathway enzymes will reduce steroid production in plants, but will not affect the formation of terpenoids derived via MEP. Equally, it is possible to inhibit terpenoid production without affecting steroid formation by the use of MEP pathway inhibitors, such as the antibiotic fosmidomycin from Streptomyces lavendulae. This acts as an analogue of the rearrangement intermediate in the reaction catalysed by MEP synthase (Figure 5.6). Enzymes of the MEP pathway are attractive targets for development of drugs against microbial diseases such as malaria or tuberculosis, since the MEP pathway is utilized by the pathogen but is not present in humans. Regulation of cholesterol production in humans is an important health concern (see page 251); the widely used statin drugs are specific inhibitors of the mevalonate pathway enzyme HMG-CoA reductase (see page 98).

HEMITERPENES (C5 ) IPP and DMAPP are reactive hemiterpene intermediates in the pathways leading to more complex terpenoid structures. They are also used as alkylating agents in the formation of meroterpenoids, as indicated above, but examples of these structures are discussed elsewhere under the section appropriate to the major substructure, e.g. alkaloids, shikimate, acetate. Relatively few true hemiterpenes are produced in nature. Isoprene, a volatile compound which is released in huge amounts by many species of plants, especially woody trees such as oaks, willows, poplars, and spruce, is the notable example. Isoprene is formed by loss of a proton from the allylic cation. Alternatively, quenching the allylic cation with water leads to methylbutenol, produced by several species of pine (Figure 5.8).

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 193 OH HO2C

OH

HO O

OP OH 1-deoxyD-xylulose 5-P

HO2C

O

OH

O

pyruvic acid

OH O

HO HO

OPP

mevalonic acid (MVA)

acetic acid

OPP

OH OH

labelling of IPP via MEP pathway

labelling of IPP via MVA pathway

[1-13C]-D-glucose

Figure 5.7

– H+ H E1

E1 allylic cation

isoprene

OPP DMAPP

+ H2O

E2

HO

E2 2-methyl-3-buten-2-ol

allylic cation E1: isoprene synthase E2: methylbutenol synthase

Figure 5.8

electrophilic addition giving tertiary cation OPP DMAPP

E1

OPP allylic cation

HR HS E1 E OPP

E1: geranyl diphosphate synthase

OPP HR H S stereospecific loss of proton

≡

OPP geranyl PP (GPP)

Figure 5.9 MONOTERPENES (C10 ) Enzyme-catalysed combination of DMAPP and IPP yields geranyl diphosphate (GPP; Figure 5.9). This is believed to involve ionization of DMAPP to the allylic cation, addition to the double bond of IPP, followed by loss of a proton. The proton lost (HR ) is stereochemically analogous to that lost on the isomerization of IPP to DMAPP;

indeed, the two reaction mechanisms are essentially the same, one involving a proton and the other a carbocation. This produces a monoterpene diphosphate, geranyl PP, in which the new double bond is trans (E). Linalyl PP and neryl PP are isomers of GPP, and are likely to be formed from GPP by ionization to the allylic cation, allowing a change in attachment of the diphosphate group (to the tertiary carbon in linalyl PP) or a

194 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition single bond in LPP allows rotation

OPP

OPP

E

OPP

Z

OPP

OPP

≡ geranyl PP (GPP)

OPP

linalyl PP (LPP)

neryl PP (NPP)

resonance-stabilized allylic cation (geranyl cation)

resonance-stabilized allylic cation (neryl cation)

Figure 5.10

OPP OPP E1: geraniol synthase E2: geraniol dehydrogenase E3: linalool synthase E4: myrcene synthase (GPP is substrate for E1, E3, E4)

OPP

GPP

LPP

NPP

OH OH

OH H O 2

geranyl cation

geraniol (geranium oil) NADP+

O

H2O E3

OH

E1

reduction citronellol (rose oil)

H 2O

NADP+

E2

neryl cation

nerol (rose oil)

− H+

E2

E4

linalool (coriander oil) CH3COSCoA OAc

O

O

reduction

citronellal (citronella oil)

tautomerism via dienol?

geranial (E-citral ) (lemon oil)

O

neral (Z-citral) (lemon oil)

β-myrcene (hops)

linalyl acetate (lavender oil)

Figure 5.11 change in stereochemistry at the double bond (to Z in neryl PP) (Figure 5.10). These three compounds, by relatively modest changes, can give rise to a range of linear monoterpenes found as components of volatile oils used

in flavouring and perfumery (Figure 5.11). The resulting compounds may be hydrocarbons, alcohols, aldehydes, or perhaps esters, especially acetates by reaction with acetyl-CoA. Where enzymes have been characterized, it

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 195 MONOCYCLIC

BICYCLIC

≡

≡

pinane type

camphane/bornane type

≡ fenchane type

menthane type

≡

≡

isocamphane type

carane type

thujane type

Figure 5.12 LPP is preferred substrate electrophilic addition giving tertiary cation OPP

OPP OPP OPP GPP

LPP

NPP

delocalized allylic cation− diphosphate ion-pair

menthyl/α-terpinyl cation

Figure 5.13 has been demonstrated that the reactions proceed through the carbocation intermediates. Thus, geraniol is the result of addition of water to the geranyl cation, and is not formed by hydrolysis of GPP. The range of monoterpenes encountered is extended considerably by cyclization reactions, and monocyclic or bicyclic systems can be created. Some of the more important examples of these ring systems are shown in Figure 5.12. A considerable amount of information about the enzymes (terpene cyclases), genes, and cyclization mechanisms is now available, providing a satisfactory and detailed picture of these natural products. Cyclizations would not be expected to occur with the precursor GPP, the E stereochemistry of the double bond being unfavourable for ring formation (Figure 5.13). Neryl PP or linalyl PP, however, do have favourable stereochemistry, and either or both of these would be more immediate precursors of the monocyclic menthane system, formation of which could be represented as shown, generating a carbocation (termed menthyl or α-terpinyl) that has the menthane skeleton. It has been found that monoterpene

cyclase enzymes are able to accept all three diphosphates, with linalyl PP being the best substrate, and it appears they have the ability to isomerize the substrates initially as well as to cyclize them. It is convenient, therefore, to consider the species involved in the cyclization as the delocalized allylic cation tightly bound to the diphosphate anion, and bond formation follows due to the proximity of the π-electrons of the double bond (Figure 5.13). In Chapter 2, the possible fates of carbocations were discussed. These include quenching with nucleophiles (especially water), loss of a proton, cyclization, and the possibility that Wagner–Meerwein rearrangements might occur (see page 15). All of these feature strongly in terpenoid biosynthesis, and examples are shown in Figures 5.14 and 5.15. The newly generated menthyl cation could be quenched by attack of water, in which case the alcohol α-terpineol would be formed, or it could lose a proton to give limonene (Figure 5.14). Alternatively, folding the cationic side-chain towards the double bond (via the surface characteristics of the enzyme) would allow a repeat of the cyclization mechanism and produce

196 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition cyclopropane ring formation

electrophilic addition creates 4-membered ring and tertiary carbocation

GPP H

menthyl/α-terpinyl cation H 2O

E1

E2

–

– H+

E4

1,2-shift creates secondary cation, but reduces ring strain

H+

car-3-ene

H

1,2-shift creates tertiary cation

W–M 1,2-alkyl shift

limonene

– H+

E5

E5

W–M 1,2-alkyl shift

fenchyl cation

pinyl cation

OH α-terpineol

electrophilic addition creates 5-membered ring and secondary carbocation

– H+

H2O

isocamphyl cation

bornyl cation

PPO−

E6

– H+

E7

+

OPP

OH

E3

OH

O cineole

E1: E2: E3: E4: E5:

α-terpineol synthase limonene synthase 1,8-cineole synthase 3-carene synthase pinene synthase

α-pinene

β-pinene

W–M = Wagner–Meerwein rearrangement E6: fenchol synthase E7: bornyl diphosphate synthase E8: bornyl diphosphate phosphohydrolase E9: borneol dehydrogenase (GPP is substrate for E1−E7)

fenchol

camphene

bornyl PP E8

O O

OH

O

O

E9 fenchone

borneol

camphor

Figure 5.14 bicyclic pinyl and bornyl cations, according to which end of the double bond was involved in forming the new bonds. Thus α-pinene and β-pinene arise by loss of different protons from the pinyl cation, producing the double bonds as cyclic or exocyclic respectively. Borneol could potentially result from quenching of the bornyl cation with water; unusually, though, this alcohol is actually derived by hydrolysis of bornyl diphosphate. Oxidation of the secondary alcohol in borneol then generates the ketone camphor. A less common termination step involving loss of a proton is also shown in Figure 5.14. This is the formation of a cyclopropane ring as exemplified by 3-carene and generation of the carane skeleton. The chemistry of terpenoid formation is essentially based on the reactivity of carbocations, even though in nature these cations may not exist as discrete species, but rather as tightly bound ion pairs with a counter anion, e.g. diphosphate. The analogy with carbocation

chemistry is justified, however, since a high proportion of natural terpenoids have skeletons which have suffered rearrangement processes. Rearrangements of the Wagner–Meerwein type (see page 15), in which carbon atoms or hydride migrate to achieve enhanced stability for the cation via tertiary against secondary character, or by reduction of ring strain, give a mechanistic rationalization for the biosynthetic pathway. The bicyclic pinyl cation, with a strained four-membered ring, rearranges to the less-strained five-membered fenchyl cation, a change which presumably more than makes up for the unfavourable tertiary to secondary carbocation transformation. This produces the fenchane skeleton, exemplified by fenchol and fenchone (Figure 5.14). The isocamphyl tertiary carbocation is formed from the bornyl secondary carbocation by a Wagner–Meerwein rearrangement, and so leads to camphene. Examples of Wagner–Meerwein rearrangements involving hydride migrations are featured in Figure 5.15.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 197

H menthyl/α-terpinyl cation W–M 1,3-hydride shift

H although the menthyl cation is tertiary, the 1,3-shift creates a resonancestabilized allylic cation

1,2-shift produces a new tertiary cation

phellandryl cation – H+

– E1

W–M = Wagner–Meerwein rearrangement

W–M 1,2-hydride shift

terpinen-4-yl cation

H+

– H+

– H+

E2

thujyl cation

H2O

– H+

E3

O oxidation + reduction

OH α-phellandrene E1: β-phellandrene synthase E2: γ-terpinene synthase

β-phellandrene

α-terpinene

γ-terpinene

terpinen-4-ol

sabinene

thujone

E3: sabinene synthase (GPP is substrate for all enzymes)

Figure 5.15 The menthyl cation, although it is a tertiary, may be converted by a 1,3-hydride shift into a favourable resonance-stabilized allylic cation. This allows the formation of α- and β-phellandrenes by loss of a proton from the phellandryl carbocation. A 1,2-hydride shift converting the menthyl cation into the terpinen-4-yl cation only changes one tertiary carbocation system for another, but allows formation of α-terpinene, γ-terpinene, and the α-terpineol isomer terpinen-4-ol. A further cyclization reaction on the terpinen-4-yl cation generates the thujane skeleton, e.g. sabinene and thujone. Terpinen-4-ol is the primary antibacterial component of tea tree oil from Melaleuca alternifolia (Myrtaceae); thujone has achieved notoriety as the neurotoxic agent in wormwood oil from Artemisia absinthium (Compositae/Asteraceae) used in preparation of the drink absinthe, now banned in most countries. So far, little attention has been given to the stereochemical features of the resultant monoterpene. Individual enzyme systems present in a particular organism will, of course, control the folding of the

substrate molecule and, thus, define the stereochemistry of the final product. Most monoterpenes are optically active, and there are many examples known where enantiomeric forms of the same compound can be isolated from different sources, e.g. (+)-camphor in sage (Salvia officinalis; Labiatae/Lamiaceae) and (−)-camphor in tansy (Tanacetum vulgare; Compositae/Asteraceae), or (+)-carvone in caraway (Carum carvi: Umbelliferae/Apiaceae) and (−)-carvone in spearmint (Mentha spicata; Labiatae/Lamiaceae). There are also examples of compounds found in both enantiomeric forms in the same organism, examples being (+)- and (−)-limonene in peppermint (Mentha × piperita; Labiatae/Lamiaceae) and (+)- and (−)-α-pinene in pine (Pinus species; Pinaceae). The individual enantiomers can produce different biological responses, especially towards olfactory receptors in the nose. Thus, the characteristic odour of caraway is due to (+)-carvone, whereas (−)-carvone smells of mint. (+)-Limonene smells of oranges, whilst (−)-limonene resembles the smell of lemons. The origins of the different enantiomeric forms of limonene and

198 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition GPP can be folded in two different ways, thus allowing generation of enantiomeric LPP molecules OPP

PPO

OPP

PPO

GPP

GPP

OPP

OPP

(–)-(3R)-LPP

OPP

OPP

(+)-(3S)-LPP

− H+

α-terpinyl cation

− H+

(+)-(R)-limonene

− H+

− H+

pinyl cation

α-terpinyl cation

(–)-(S)-limonene

(+)-α-pinene

(–)-α-pinene

pinyl cation

Figure 5.16 α-pinene are illustrated in Figure 5.16. This shows the precursor GPP being folded in two mirror-image conformations, leading to formation of the separate enantiomers of linalyl PP. Analogous carbocation reactions will then explain production of the optically active monoterpenes. Where a single plant produces both enantiomers, it appears to contain two separate enzyme systems each capable of elaborating a single enantiomer. Furthermore, a single enzyme typically accepts GPP as substrate, catalyses the isomerization to linalyl PP, and converts this into a final product without the release of free intermediates. Terpenoid cyclase enzymes, even highly pure proteins obtained by gene expression, rarely convert their substrate into a single product. Sometimes, multiple products in varying amounts, e.g. limonene, myrcene, α-pinene, and β-pinene, are synthesized by a single enzyme,

reflecting the common carbocation chemistry involved in these biosyntheses. This suggests that the enzyme is predominantly providing a suitable environment for the folding and cyclization of the substrate, whilst carbocation chemistry is responsible for product formation. Many of the transformations included in Figures 5.14 and 5.15 are based on analysis of these alternative products and how carbocation chemistry interrelates them. Subsequent reactions, such as oxidation of an alcohol to a ketone, e.g. borneol to camphor (Figure 5.14), or heterocyclic ring formation in the conversion of α-terpineol into cineole (Figure 5.14), require additional enzyme systems. In other systems, a particular structure may be found as a mixture of diastereoisomers. Peppermint (Mentha × piperita; Labiatae/Lamiaceae) typically produces (−)-menthol, with smaller amounts of the stereoisomers (+)-neomenthol, (+)-isomenthol, and

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 199 oxidation HO allylic hydroxylation

NAD+

O

E2

O2 NADPH E1 (–)-trans-carveol E3

allylic hydroxylation

reduction allylic isomerization

oxidation

O2 NADPH (–)-limonene

(–)-carvone

NAD+

E6

OH E4

E5

O

O

NADPH (–)-piperitone

O NADPH E6

(–)-transisopiperitenol

(–)-isopiperitenone

piperitenone

reduction

O OH

NADPH E1: (−)-limonene 6-hydroxylase E2: (−)-trans-carveol dehydrogenase E3: (−)-limonene 3-hydroxylase E4: (−)-trans-isopiperitenol dehydrogenase E5: (+)-cis-isopulegone isomerase E6: (+)-pulegone reductase E7: (−)-isopiperitenone reductase E8: (−)-menthone:(+)-neomenthol reductase E9: (−)-menthone:(−)-menthol reductase E10: (+)-menthofuran synthase

(+)-piperitone reduction

E7

NADPH (+)-neomenthol

reduction

E8 NADPH

reduction allylic isomerization

E9

O

NADPH E6

(–)-menthone

E5

O

O

(–)-menthol (levomenthol)

NADPH reduction

E6

(+)-cisisopulegone

(+)-pulegone

NADPH

reduction O E10

allylic hydroxylation

OH

(+)-isomenthol

(+)-isomenthone NADPH

E9

reduction

OH O

E8

O2 NADPH

hemiketal formation

− H2O O

OH

reduction

O

OH

OH (+)-menthofuran

(+)-neoisomenthol

Figure 5.17 (+)-neoisomenthol, covering four of the possible eight stereoisomers (Figure 5.17). Oils from various Mentha species also contain significant amounts of ketones, e.g. (−)-menthone, (+)-isomenthone, (−)-piperitone, or (+)-pulegone. The metabolic relationship of these various compounds has been established as in Figure 5.17, which illustrates how the stereochemistry at each centre can be determined by stereospecific reduction processes on double bonds or carbonyl groups. Note that some of the enzymes involved have rather broad substrate

specificity. The pathway also exemplifies that oxygen functions can be introduced into the molecule at positions activated by adjacent double bonds (allylic oxidation), as well as being introduced by quenching of carbocations with water. These reactions are catalysed by cytochrome P-450-dependent monooxygenases. Thus, limonene is a precursor of carvone (the main constituent of spearmint oil from Mentha spicata) as well as menthone and piperitone, where initial hydroxylation occurs at an alternative allylic site on the ring. Menthofuran exemplifies a

Citrus aurantium ssp. bergamia (Rutaceae)

Cinnamomum camphora (Lauraceae)

Carum carvi (Umbelliferae /Apiaceae) Elettaria cardamomum (Zingiberaceae)

Bergamot

Camphor oil

Caraway

Cymbopogon winterianus, C. nardus (Graminae/Poaceae)

Coriandrum sativum (Umbelliferae /Apiaceae)

Anethum graveolens (Umbelliferae /Apiaceae) Eucalyptus globulus, E. smithii, E. polybractea (Myrtaceae) Eucalyptus citriodora (Myrtaceae)

Citronella

Coriander

Dill

Eucalyptus (lemon-scented)

Eucalyptus

Chamaemelum nobile (Anthemis nobilis) (Compositae/ Asteraceae)

Chamomile (Roman chamomile)

Cardamom

Plant source

Oil

fresh leaves

fresh leaves

ripe fruit

ripe fruit

fresh leaves

0.8

1–3

3–4

0.3–1.8

0.5–1.2

0.4–1.5

3–7

ripe fruit

dried flowers

3–7

1–3

0.5

Oil content (%)

ripe fruit

wood

fresh fruit peel (expression)

Plant part used

Table 5.1 Volatile oils (ii): containing principally terpenoid compounds

cineole (= eucalyptol) (70–85) α-pinene (14) citronellal (65–85)

α-terpinyl acetate (25–35) cineole (25–45) linalool (5) aliphatic esters of angelic, tiglic, isovaleric, and isobutyric acids (75–85) small amounts of monoterpenes (+)-citronellal (25–55) geraniol (20–40) (+)-citronellol (10–15) geranyl acetate (8) (+)-linalool (60–75) γ-terpinene (5) α-pinene (5) camphor (5) (+)-carvone (40–65)

limonene (42) linalyl acetate (27) γ-terpinene (8) linalool (7) camphor (27–45) cineole (4–21) safrole (1–18) (+)-carvone (50–70) limonene (47)

Major constituents with typical (%) composition

perfumery

flavour, antiseptic, aromatherapy

flavour, carminative

flavour, carminative

perfumery, aromatherapy, insect repellent

flavouring, aromatherapy; blue colour of oil is due to chamazulene

flavour, carminative; ingredient of curries, pickles

flavour, carminative, aromatherapy

flavouring, aromatherapy, perfumery; also contains the furocoumarin bergapten (up to 5%) and may cause severe photosensitization (see page 165) soaps

Uses, notes

200 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Lavandula angustifolia, L. officinalis (Labiatae/Lamiaceae) Citrus limon (Rutaceae)

Cymbopogon citratus (Graminae/Poaceae) Matricaria chamomilla (Chamomilla recutica) (Compositae /Asteraceae) Citrus aurantium ssp. amara (Rutaceae)

Lavender

Lemon-grass

Orange (sweet)

Orange (bitter)

Matricaria (German chamomile)

Citrus sinensis (Rutaceae)

Juniperus communis (Cupressaceae)

Juniper

Lemon

Zingiber officinale (Zingiberaceae)

Ginger

dried fruit peel (expression)

dried fruit peel (expression)

dried flowers

fresh leaves

dried fruit peel (expression)

fresh flowering tops

dried ripe berries

dried rhizome

0.3

0.5–2.5

0.3–1.5

0.1–0.3

0.1–3

0.3–1

0.5–2

1.5–3

(+)-limonene (90–95) myrcene (2)

(+)-limonene (60–80) β-pinene (8–12) γ-terpinene (8–10) citral (= geranial + neral) (2–3) citral (= geranial + neral) (50–85) (−)-α-bisabolol (10–25) bisabolol oxides A and B (10–25) chamazulene (1–15) (+)-limonene (92–94) myrcene (2)

zingiberene (34) β-sesquiphellandrene (12) β-phellandrene (8) β-bisabolene (6) α-pinene (45–80) myrcene (10–25) limonene (1–10) sabinene (0–15) linalyl acetate (25–45) linalool (25–38)

(continued overleaf )

flavouring, aromatherapy; the main flavour and odour come from the minor oxygenated components; terpeneless orange oil is obtained by removing much of the terpenes under reduced pressure; this oil contains about 20% aldehydes, mainly decanal flavouring, aromatherapy; the main flavour and odour come from the minor oxygenated components; terpeneless orange oil is obtained by removing much of the terpenes under reduced pressure; this oil contains about 20% aldehydes, mainly octanal and decanal

flavouring; dark blue colour of oil is due to chamazulene

perfumery, aromatherapy; inhalation produces mild sedation and facilitates sleep flavouring, perfumery, aromatherapy; terpeneless lemon oil is obtained by removing much of the terpenes under reduced pressure; this oil is more stable and contains 40–50% citral perfumery, aromatherapy

flavouring, antiseptic, diuretic, aromatherapy; juniper berries provide the flavouring for gin

flavouring; the main pungent principals (gingerols) in ginger are not volatile (see page 168)

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 201

Pinus palustris or other Pinus species (Pinaceae) Pinus mugo ssp. pumilio (Pinaceae)

Pine

Rosa damascena, R. gallica, R. alba, and R. centifolia (Rosaceae)

Rosmarinus officinalis (Labiatae/Lamiaceae)

Salvia officinalis (Labiatae/Lamiaceae)

Santalum album (Santalaceae)

Mentha spicata (Labiatae/Lamiaceae)

Rose (attar of rose, otto of rose)

Rosemary

Sage

Sandalwood

Spearmint

Pumilio pine

fresh leaf

Mentha × piperita (Labiatae/Lamiaceae)

Peppermint

fresh leaf

heartwood

fresh flowering tops

fresh flowering tops

fresh flowers

needles

needles, twigs

fresh flowers

Citrus aurantium ssp. amara (Rutaceae)

Orange flower (Neroli)

Plant part used

Plant source

Oil

Table 5.1 (continued)

1–2

4.5–6.3

0.7–2.5

1–2

0.02–0.03

0.3–0.4

1–3

0.1

Oil content (%)

(−)-carvone (50–70) (−)-limonene (2–25)

α- and β-phellandrene (60) α- and β-pinene (10–20) bornyl acetate (3–10) citronellol (36) geraniol (17) 2-phenylethanol (3) C14 –C23 straight-chain hydrocarbons (25) cineole (15–45) α-pinene (10–25) camphor (10–25) β-pinene (8) thujone (40–60) camphor (5–22) cineole (5–14) β-caryophyllene (10) limonene (6) sesquiterpenes: α-santalol (50) β-santalol (21)

linalool (36) β-pinene (16) limonene (12) linalyl acetate (6) menthol (30–50) menthone (15–32) menthyl acetate (2–10) menthofuran (1–9) α-terpineol (65)

Major constituents with typical (%) composition

flavouring, carminative, aromatherapy

perfumery, aromatherapy

aromatherapy, food flavouring

perfumery, aromatherapy

inhalant; the minor components bornyl acetate and borneol are mainly responsible for the aroma perfumery, aromatherapy

antiseptic, disinfectant, aromatherapy

flavouring, carminative, aromatherapy

flavour, perfumery, aromatherapy

Uses, notes

202 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Thymus vulgaris (Labiatae/Lamiaceae)

Pinus palustris and other Pinus spp. (Pinaceae)

Thyme

Turpentine oil

distillation of the resin (turpentine) secreted from bark

fresh flowering tops

fresh leaf

0.5–2.5

1.8

terpinen-4-ol (30–45) γ-terpinene (10–28) α-terpinene (5–13) p-cymene (0.5–12) cineole (0.5–10) α-terpineol (1.5–8) thymol (40) p-cymene (30) linalool (7) carvacrol (1) (+)- and (−)-α-pinene (35:65) (60–70) β-pinene (20–25)

counter-irritant, important source of industrial chemicals; the residue from distillation is colophony (rosin), composed chiefly of diterpene acids (abietic acids, see page 230)

antiseptic, aromatherapy, food flavouring

antiseptic, aromatherapy; an effective broad-spectrum antiseptic widely used in creams, cosmetics, toiletries

Note: Major volatile oils have been divided into two groups. Those oils containing principally chemicals which are terpenoid in nature and which are derived by the methylerythritol phosphate or mevalonate pathways are given in this table. Oils which are composed predominantly of aromatic compounds which are derived via the shikimate pathway are listed in Table 4.1 on page 158. The remarks in the footnotes to Table 4.1 are also applicable here. Structures of terpenoids are shown in Figures 5.11–5.18.

Melaleuca alternifolia (Myrtaceae)

Tea tree

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 203

204 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

HO regular monoterpene skeleton

OH

p-cymene

thymol

carvacrol

Figure 5.18 further cytochrome P-450-dependent oxidative modification that leads to generation of a heterocyclic furan ring via the hemiketal. Both pulegone and menthofuran are considered hepatotoxic. Pulegone is a major constituent of oil of pennyroyal from Mentha pulegium, which has a folklore history as an abortifacient. Pulegone is metabolized in humans first to menthofuran, by the same mechanism as it is in the plant pathway, and then to electrophilic metabolites that form adducts with cellular proteins (compare pyrrolizidine alkaloids, page 325). High menthofuran levels in peppermint oils are regarded as undesirable. Peppermint plants transformed with an antisense version of the menthofuran synthase gene produce less than half the normal amounts of this metabolite. p-Cymene and the phenol derivatives thymol and carvacrol (Figure 5.18), found in thyme (Thymus vulgaris; Labiatae/Lamiaceae), are representatives of a small group of aromatic compounds that are produced in nature from isoprene units, rather than by the much more common routes to aromatics involving acetate or shikimate (see also cannabinol, page 119, and gossypol, page 220). These compounds all possess the carbon skeleton typical of monocyclic monoterpenes, and their structural relationship to limonene and the more common oxygenated monoterpenes, such as menthone or carvone, suggests pathways in which additional dehydrogenation reactions are involved. Data on volatile oils containing terpenoid constituents isolated from these and other plant materials are given in Table 5.1. Volatile oils in which the main components are aromatic and derived from the shikimate pathway are listed in Table 4.1, page 158. IRREGULAR MONOTERPENES A number of natural monoterpene structures contain carbon skeletons which, although obviously derived from two isoprene C5 units, do not seem to fit the regular head-to-tail coupling mechanism, e.g. those in Figure 5.19. These structures are termed irregular

irregular monoterpene skeletons

Figure 5.19 monoterpenes and seem to be limited almost exclusively to members of the Compositae/Asteraceae plant family. Allowing for possible rearrangements, the two isoprene units appear to have coupled in another manner, and this is borne out by information available on their biosynthesis, though this is far from complete. Thus, although DMAPP and IPP are utilized in their biosynthesis, GPP and neryl PP do not appear to be involved. Pre-eminent amongst these structures are chrysanthemic acid and pyrethric acid (Figure 5.20), found in ester form as the pyrethrins (pyrethrins, cinerins, and jasmolins, Figure 5.20), which are valuable insecticidal components in pyrethrum flowers, the flower heads of Chrysanthemum cinerariaefolium (Compositae/Asteraceae) [Box 5.1]. These cyclopropane structures are readily recognizable as derived from two isoprene units, and a mechanism for the derivation of chrysanthemic acid via chrysanthemyl diphosphate is given in Figure 5.21. This invokes two DMAPP units joining by a modification of the standard mechanism, with termination achieved by cyclopropane ring formation (compare carene, page 196). This mechanism is seen again later; it is identical to that involved in the formation of presqualene PP during steroid biosynthesis (see page 235) and of prephytoene PP in carotenoid formation (see page 300). Relatively little is known about the origins of pyrethrolone, cinerolone, and jasmolone (Figure 5.20), the alcohol portions of the pyrethrins, though it is likely that these are cyclized and modified fatty acid derivatives, the cyclization process resembling the biosynthetic pathway to prostaglandins (see page 60). Thus, α-linolenic acid via 12-oxophytodienoic acid could be the precursor of jasmolone, with β-oxidation and then decarboxylation accounting for the chain shortening (Figure 5.22). Certainly, this type of pathway operates in the formation of jasmonic acid (Figure 5.22), which forms part of a general signalling system in plants, particularly the synthesis of secondary metabolites in response to wounding or microbial infection.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 205

CO2H

O

CO2H MeO2C

chrysanthemic acid

R1

pyrethric acid

R2

O O

HO

HO

R1

HO

O pyrethrolone

O cinerolone

O jasmolone

R2

pyrethrin I

Me

CH=CH2

cinerin I

Me

Me

jasmolin I

Me

Et

pyrethrin II

CO2Me

CH=CH2

cinerin II

CO2Me

Me

jasmolin II

CO2Me

Et

Figure 5.20 DMAPP

electrophilic addition giving tertiary cation

hydrolysis of phosphate ester; oxidation of alcohol to acid

H

OPP DMAPP

loss of proton via cyclopropyl ring formation OPP

OPP

E1

CO2H

E1

chrysanthemic acid

chrysanthemyl PP E1: chrysanthemyl diphosphate synthase (substrate DMAPP)

Figure 5.21 radical oxidation and formation of peroxide (compare Figure 3.23) O

CO2H

H

O O

α-linolenic acid

CO2H

nonenzymic

CO2H

CO2H

E1 O

E2

O

OH E3

CO2H

b-oxidation

HO CO2H

?

E4 O

jasmonic acid E1: lipoxygenase E2: allene oxide synthase

O

7-iso-jasmonic avid

O

12-oxophytodienoic acid

O

jasmolone

E3: allene oxide cyclase E4: 12-oxophytodienoate reductase

Figure 5.22

Box 5.1 Pyrethrins The pyrethrins are valuable insecticidal components of pyrethrum flowers, Chrysanthemum cinerariaefolium (=Tanacetum cinerariifolium) (Compositae/Asteraceae). The flowers are harvested just before they are fully expanded, and usually processed to an extract. Pyrethrum cultivation is conducted in East Africa, especially Kenya, and more recently in Ecuador and Australia. The natural pyrethrins are used as a constituent of insect sprays for household use and as a post-harvest insecticides, having a

206 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.1 (continued) rapid action on the nervous system of insects, whilst being biodegradable and non-toxic to mammals, though they are toxic to fish and amphibians. This biodegradation, initiated by air and light, means few insects develop resistance to the pyrethrins, but it does limit the lifetime of the insecticide under normal conditions to just a few hours. The flowers may contain 0.7–2% of pyrethrins, representing about 25–50% of the extract. A typical pyrethrin extract contains pyrethrin I (35%), pyrethrin II (32%), cinerin I (10%), cinerin II (14%), jasmolin I (5%), and jasmolin II (4%), which structures represent esters of chrysanthemic acid or pyrethric acid with the alcohols pyrethrolone, cinerolone, and jasmolone (Figure 5.20). Pyrethrin I is the most insecticidal component, with pyrethrin II providing much of the rapid knock-down (paralysing) effect. A wide range of synthetic pyrethroid analogues, e.g. bioresmethrin, tetramethrin, phenothrin, permethrin, cypermethrin, and deltamethrin (Figure 5.23), have been developed which have increased lifetimes up to several days and greater toxicity towards insects. These materials have become widely used household and agricultural insecticides; the commercial insecticides are often a mixture of stereoisomers. Tetramethrin, bioresmethrin, and phenothrin are all esters of chrysanthemic acid, but with a modified alcohol portion, providing improvements in knock-down effect and in insecticidal activity. Replacement of the terminal methyl groups of chrysanthemic acid with halogen atoms, e.g. permethrin, conferred greater stability towards air and light and opened up the use of pyrethroids in agriculture. Inclusion of a cyano group in the alcohol portion, as in cypermethrin and deltamethrin, improved insecticidal activity several-fold. Modern pyrethroids now have insecticidal activities over a thousand times that of pyrethrin I, whilst maintaining extremely low mammalian toxicity. Permethrin and phenothrin are employed against skin parasites such as head lice in humans.

O O O

N

O

O

O O bioresmethrin Cl

O O

Cl O

phenothrin

tetramethrin

Br

O RS R O

O O

Br

O R = H, permethrin R = CN, cypermethrin

CN

O deltamethrin

Figure 5.23

IRIDOIDS (C10 ) The iridane skeleton (Figure 5.24) found in iridoids is monoterpenoid in origin and contains a cyclopentane ring which is usually fused to a six-membered oxygen heterocycle. The iridoid system arises from geraniol by a type of folding (Figure 5.25) which is different from that already encountered with monoterpenoids; also markedly different is the lack of phosphorylated intermediates and subsequent carbocation mechanism in its formation. The fundamental cyclization to iridodial is formulated as attack of hydride on the dialdehyde, produced by a series of hydroxylation and oxidation reactions on geraniol. Further oxidation gives iridotrial, in which hemiacetal formation then leads to production of the heterocyclic ring. In

O iridane

iridoid

secoiridoid

O iridoid

Figure 5.24

O

O secoiridoid

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 207 cyclization formulated as initiated by electrophilic addition utilizing the unsaturated carbonyl, terminated by addition of hydride; the imineassisted mechanism shown below is more realistic

GPP E1 oxidations

O2 NADPH OH

H

NADPH

OH

E2

CHO

E3

E4

H

H

H

O

CHO

H

H

OH

CHO

O 10-oxogeranial

OH 10-hydroxygeraniol

geraniol

hemiacetal formation

H

iridodial (enol form)

iridodial (keto form)

OH

O

iridodial (hemiacetal form)

O (NADPH) H

hemiacetal formation

CHO

CHO

H OHC

HN

formation of alkene and ring cleavage

Enz

Fe O

H HO

H H

H

Enz

HO

H

H H

OGlc

O

MeO2C

HO

Fe

E7

OH

HO H

H MeO2C

O

H MeO2C

OGlc

O

loganic acid

O

OGlc

– H2O

CHO

H

H

OGlc

O

MeO2C

O2 NADPH

H H

E5 HO2C

OGlc

O

deoxyloganic acid glucosylation has now transformed the hemiacetal into an acetal

O2 NADPH

OH H

OGlc

H

SAM E6 H HO2C

loganin Enz

O

HO

OGlc

O

MeO2C

OH

oxidation of aldehyde to acid; glucosylation HO

H

H

iridotrial (hemiacetal form)

iridotrial (keto form)

HN

H OHC

CHO

oxidation leading to radical H

OGlc

O

MeO2C

H

Enz

CHO

tryptamine

terpenoid indole alkaloids

secologanin E1: geraniol synthase E2: geraniol 10-hydroxylase E3: 10-hydroxygeraniol oxidoreductase (acyclic monoterpene primary alcohol dehydrogenase)

E4: E5: E6: E7:

monoterpene cyclase 7-deoxyloganin 7-hydroxylase (also accepts acid as substrate) loganic acid methyltransferase secologanin synthase

Figure 5.25 iridotrial, there is an equal chance that the original methyl groups from the head of geraniol end up as the aldehyde or in the heterocyclic ring. A large number of iridoids are found as glycosides, e.g. loganin; glycosylation effectively transforms the hemiacetal linkage into an acetal. The pathway to loganin involves, in addition, a sequence of reactions in which the remaining aldehyde group is

oxidized to the acid and methylated, and the cyclopentane ring is hydroxylated. Loganin is a key intermediate in the pathway to a range of complex terpenoid indole alkaloids (see page 369) and tetrahydroisoquinoline alkaloids (see page 363). Fundamental in this further metabolism is cleavage of the simple monoterpene skeleton, which is still recognizable in

208 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition geraniol

H

≡

O H

HO

H H H

H

OH O H

O

≡

O H

nepetalactone

iridodial

O H

O

O

OH

O

CO2Me

HO O

O

OGlc

oleuropin

11

7

CO2H

H

6 8

H

4 5 9 1

H 10

CO2Me

HO2C

H

CO2Me

O

O H

3

O2

HO

O H

OGlc

deoxyloganic acid

O

CO2Me

HO O

OGlc

OGlc oleoside 11-methyl ether

7-epi-loganin

OGlc

ligstroside

geraniol

H

H O

H

OH

8-epi-iridodial

CO2H

− CO2

O H

HO

HO OH

OH

O HO

OGlc 8-epi-deoxyloganic acid

O H

O

OGlc

harpagide

O H

OGlc

harpagoside

Figure 5.26 loganin, to give secologanin, representative of the secoiridoids (Figure 5.24). This is catalysed by a cytochrome P-450-dependent monooxygenase, and a radical mechanism is proposed in Figure 5.25. Secologanin now contains a free aldehyde group, together with further aldehyde and enol groups, with these latter two fixed as an acetal by the presence of the glucose. As we shall see with some of the complex alkaloids, these functionalities can be released again by hydrolysing off the glucose and reopening the hemiacetal linkage. Well over a thousand different natural iridoids and secoiridoids are known. Structural variation arises predominantly from hydroxylations, esterifications, and changes in stereochemistry. Another major change is the loss of a carbon atom by a decarboxylation mechanism. A few examples showing their relationship to the intermediates of Figure 5.25 are presented in Figure 5.26.

Nepetalactone from catmint Nepeta cataria (Labiatae/Lamiaceae), a powerful attractant and stimulant for cats, is produced from iridodial by simple oxidation of the hemiacetal to a lactone (Figure 5.26). Ligstroside and oleuropin are phenolic esters found in olive oil (see page 47) from Olea europea (Oleaceae), and are believed to contribute to the health benefits of this oil. These compounds are secoiridoids, but are formed from the 7-epimer of loganin; in this case, the stereochemistry of 7-hydroxylation is different from the normal loganin pathway. Harpagide and harpagoside are examples of decarboxylated iridoids; these particular examples are formed via 8-epi-iridodial, in which geraniol is cyclized by a stereochemically different mechanism. Harpagoside is the cinnamoyl ester of harpagide. These compounds are found in devil’s claw (Harpagophytum procumbens; Pedaliaceae) and contribute to the anti-inflammatory and analgesic properties associated with this plant drug [Box 5.2].

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 209

Box 5.2 Devil’s Claw Devil’s claw is the common name for Harpagophytum procumbens (Pedaliaceae) from the appearance of its fruit, which have curved, sharp hooks. The plant is a weedy, perennial, tuberous plant with long creeping stems found in southern Africa (South Africa, Namibia, Botswana); commercial material is collected from the wild, but is often a mixture from two species: H. procumbens and H. zeyheri. Preparations of the secondary roots have gained a reputation as an anti-inflammatory and antirheumatic agent to relieve pain and inflammation in people with arthritis and similar disorders. Clinical studies appear to support its medicinal value as an anti-inflammatory and analgesic, though some findings are less positive. The main constituents of devil’s claw root are a group of decarboxylated iridoid glycosides (about 3%), including harpagoside (at least 1.2%) as the main component and smaller amounts of procumbide, harpagide, and 8-(4-coumaroyl)harpagide (Figure 5.27). The latter compound appears representative of H. zeyheri only, so indicates if this species is present in the sample. The secondary roots contain significantly higher levels of iridoids than the primary tubers. Harpagoside and related iridoids have been shown to inhibit thromboxane biosynthesis (see page 65), which may relate to the observed anti-inflammatory activity of devil’s claw. HO

HO

OH

HO

OH

OH

O O R

O H

HO OGlc

O H

OGlc

harpagide

O

O H

OGlc

procumbide

R = H, harpagoside R = OH, 8-(4-coumaroyl)harpagide

Figure 5.27

A range of epoxyiridoid esters has been identified in the drug valerian (Valeriana officinalis; Valerianaceae) [Box 5.3]. These materials, responsible for the sedative activity of the crude drug, are termed valepotriates. Valtrate (Figure 5.28) is a typical example, and

illustrates the structural relationship to loganin, though these compounds contain additional ester functions, frequently isovaleryl. The hemiacetal is now fixed as an ester, rather than as a glycoside.

Box 5.3 Valerian Valerian root consists of the dried underground parts of Valeriana officinalis (Valerianaceae), a perennial herb found throughout Europe. Drug material comes from wild and cultivated plants, and is carefully dried at low temperature (less than 40◦ C) to minimize decomposition of constituents. Valerian preparations are widely used as herbal tranquillizers to relieve nervous tension, anxiety, and as a mild sedative to promote sleep; the drug was especially popular during the First World War, when it was used to treat shell-shock. The drug does possess mild sedative and anxiolytic properties, but the roots need to be freshly harvested and carefully dried for maximum activity. The major active principles are generally held to be a number of epoxyiridoid esters called valepotriates (0.5–1.6%), the principal component of which is valtrate (about 80%) (Figure 5.28). Minor valepotriates have the same parent iridoid alcohol as valtrate, but differ with respect to esterifying acids, e.g. isovaltrate and acevaltrate (Figure 5.28), or are based on the reduced iridoid seen in didrovaltrate, again with various ester functionalities. Acid entities characterized in this group of compounds are mainly isovaleric (3-methylbutyric) and acetic (as in valtrate/isovaltrate/didrovaltrate), though more complex diester groups involving 3-acetoxyisovaleric and isovaleroxyisovaleric acids are encountered. During drying and storage, some of the valepotriate content may decompose by hydrolysis to liberate quantities of isovaleric acid, giving a characteristic odour, and structures such as baldrinal (Figure 5.28) (from valtrate) and homobaldrinal (from isovaltrate). Samples of old or poorly prepared valerian may contain negligible amounts of valepotriates. Standardized mixtures of valepotriates are available in some countries. These materials are usually extracted from the roots of other species of Valeriana which produce higher amounts

210 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.3 (continued) O

O

O

H

O

O

O

O O

O

H

O

O

O O

O

CHO

O

O

O

valtrate

CHO

O

O

baldrinal

isovaltrate

O OAc

O

O

O

O

O

H O O O

H

O

O acevaltrate

H

O O

O O

H

O

homobaldrinal

O

O

O

didrovaltrate

CO2H

O valeranone

valerenic acid

Figure 5.28 of valepotriates than V. officinalis, e.g. V. mexicana contains up to about 8%. Some other species of Valeriana which contain similar valepotriate constituents are used medicinally, including V. wallichi (Indian valerian) and V. edulis (Mexican valerian). Despite the information given above, many workers believe the sedative activity of valerian cannot be due to the valepotriates, which are rather unstable and not water soluble. Some of the sedative activity is said to arise from sesquiterpene derivatives such as valerenic acid (about 0.3%) and those found in the volatile oil content (0.5–1.3%), e.g. valeranone (Figure 5.28), which have been shown to be physiologically active. GABA and glutamine have also been identified in aqueous extracts of valerian, and these have been suggested to contribute to the sedative properties. The valepotriates valtrate and didrovaltrate are reported to be cytotoxic in vitro, and this may restrict future use of valerian. The reactive epoxide group is likely to be responsible for these cytotoxic properties.

SESQUITERPENES (C15 ) Sesquiterpenes are formed from three C5 units; the terminology comes from the Latin prefix sesqui: ‘one and a half times’. Though the mechanisms involved in their formation closely parallel those seen for the monoterpenes, sesquiterpenes are generally synthesized via the mevalonate pathway (see page 192) rather than from MEP. Addition of a further C5 IPP unit to GPP in an extension of the GPP synthase reaction (Figure 5.9) leads to the fundamental sesquiterpene precursor farnesyl diphosphate (FPP) (Figure 5.29). Again, an initial ionization of GPP seems likely, and the proton lost from C-2 of IPP is stereochemically analogous to that lost in the previous isoprenylation step. FPP can then give rise to linear and cyclic sesquiterpenes. Because of the increased chain length and an additional double bond, the number of possible cyclization modes is increased, and a huge range of mono-, bi-, and tri-cyclic structures can result; the number of known natural sesquiterpenes greatly exceeds that of known natural

OPP geranyl PP E1

OPP HR H S electrophilic addition giving tertiary cation

allylic cation

OPP E1

HR HS

stereospecific loss of proton OPP

farnesyl PP (FPP) E1: farnesyl diphosphate synthase

Figure 5.29

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 211 E

E OPP E,E-farnesyl cation

E,E-FPP

E,Z-farnesyl cation

E E,Z-FPP

nerolidyl cation

OPP

Z OPP

nerolidyl PP

Figure 5.30 monoterpenes. The stereochemistry of the double bond nearest the diphosphate can adopt an E configuration (as in FPP) or a Z configuration via ionization as found with GPP/neryl PP (Figure 5.30). In some systems, the tertiary diphosphate nerolidyl PP (compare linalyl PP, page 194) has been implicated as a more immediate precursor than FPP (Figure 5.30). This allows different possibilities for folding the carbon chain, dictated of course by the enzyme involved, and cyclization by electrophilic attack onto an appropriate double bond. As with the monoterpenes, standard reactions of carbocations rationally explain most of the common structural skeletons encountered, and a small but representative selection of these is given in Figure 5.31; sesquiterpene cyclase enzymes typically synthesize a major product accompanied by a range of related structures. One of these cyclized systems, the bisabolyl cation, is analogous to the monoterpene menthane system, and further modifications in the six-membered ring can take place to give essentially monoterpene variants with an extended hydrocarbon substituent, e.g. γ-bisabolene (Figure 5.32), which contributes to the aroma of ginger (Zingiber officinale; Zingiberaceae) along with obviously related structures such as zingiberene and β-sesquiphellandrene (Figure 5.33). Sesquiterpenes will, in general, be less volatile than monoterpenes. Simple quenching of the bisabolyl cation with water leads to α-bisabolol (Figure 5.32), a major component of matricaria (German chamomile) flowers (Matricaria chamomilla; Compositae/Asteraceae) [Box 5.5]. So-called bisabolol oxides A and B are also present, compounds probably derived from bisabolol by cyclization reactions (Figure 5.32) on an intermediate epoxide (compare Figure 4.34, page 164). Other cyclizations in Figure 5.31 lead to ring systems larger than six carbon atoms, and 7-, 10-, and

11-membered rings can be formed as shown. The two 10-membered ring systems (germacryl and cis-germacryl cations), or the two 11-membered systems (humulyl and cis-humulyl cations), differ only in the stereochemistry associated with the double bonds. However, this affects further cyclization processes and is responsible for extending the variety of natural sesquiterpene derivatives. The germacryl cation, without further cyclization, is a precursor of the germacrane class of sesquiterpenes, as exemplified by costunolide (Figure 5.34), a bitter principle found in the roots of chicory (Cichorium intybus; Compositae/Asteraceae). Costunolide is actually classified as a germacranolide, the suffix ‘olide’ referring to the lactone group (compare macrolide, page 68). The antimigraine agent in feverfew (Tanacetum parthenium; Compositae/Asteraceae) is parthenolide [Box 5.4], an epoxide derivative of costunolide (Figure 5.34). At present it is not known whether epoxidation of the double bond in costunolide is involved, or whether an earlier intermediate in the pathway is oxidized. The α,β-unsaturated carbonyl functionality seen in costunolide and parthenolide is a common feature of many of the biologically active terpenoids. The activity frequently manifests itself as a toxicity, especially cytotoxicity, as seen with the germacranolide elephantopin (Figure 5.33) from Elephantopus elatus (Compositae/Asteraceae), or skin allergies, as caused by the pseudoguaianolide (a rearranged guaianolide) parthenin (Figure 5.33) from Parthenium hysterophorus (Compositae/Asteraceae), a highly troublesome weed in India. These compounds can be considered as powerful alkylating agents by a Michael-type addition of a suitable nucleophile, e.g. thiols, onto the α,β-unsaturated carbonyl system. Such alkylation reactions are believed to explain

212 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

i

H guaiyl cation e.g. matricin, thapsigargin H

E

10

germacryl cation e.g. parthenolide

E

E,E-farnesyl cation

ii

eudesmyl cation e.g. α-santonin

11

humulyl cation e.g. humulene E

caryophyllyl cation e.g. caryophyllene

≡

W–M 1,3-hydride shift

E H

bisabolyl cation e.g. bisabolene, α-bisabolol

nerolidyl cation

amorphyl cation e.g. artemisinic acid

a 6 a E

7 a

Z

b

E,Z-farnesyl cation

carotyl cation W–M 1,3-hydride shift

b 10 H b 11

n

cis-germacryl cation

n = ring size generated cis-humulyl cation

Figure 5.31

cadinyl cation e.g. α-cadinene

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 213 H

H – H+

– H+ OH

H

E1

O γ-bisabolene

bisabolyl cation

(–)-β-bisabolene – H+

OH

OH

H

H

(–)-α-bisabolol oxide B

b

H2O

E1

OH

H

H a

O

b a (–)-α-bisabolene

O

(–)-α-bisabolol oxide A E1: γ-bisabolene synthase (FPP is substrate)

(–)-α-bisabolol

Figure 5.32

O

H

H

HO

O

O O

O O O

(–)-zingiberene

(–)−β-sesquiphellandrene

O O

O O elephantopin

O

O O

H

O O OH OH

O O thapsigargin

O

O

parthenin

O

CO2H angelic acid

α-santonin

O

CO2H tiglic acid

CO2H O

CO2H isovaleric acid

isobutyric acid

Figure 5.33 biological activity; indeed, activity is typically lost if either the double bond or the carbonyl group is chemically reduced. In some structures, additional electrophilic centres offer further scope for alkylation reactions. In parthenolide (Figure 5.35), an electrophilic epoxide group is also present, allowing transannular cyclization and generation of a second alkylation site. Cytotoxic

agents may irreversibly alkylate critical enzymes that control cell division, whilst allergenic compounds may conjugate with proteins to form antigens which trigger the allergic response. The beneficial effects of parthenolide and structurally related compounds in feverfew have been demonstrated to relate to alkylation of thiol groups.

214 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.4 Feverfew Feverfew is a traditional herbal remedy for the relief of arthritis, migraine, toothache, and menstrual difficulties. The plant is a perennial, strongly aromatic herb of the Compositae/Asteraceae family, and has been classified variously as Tanacetum parthenium (which is currently favoured), Chrysanthemum parthenium, Leucanthemum parthenium, or Pyrethrum parthenium. Studies have confirmed that feverfew is an effective prophylactic treatment for migraine in about 70% of sufferers. It reduces the frequency and severity of attacks and the vomiting associated with them. The herb has been shown to inhibit blood platelet aggregation, the release of 5-hydroxytryptamine (5-HT, serotonin) from platelets, the release of histamine from mast cells, and the production of prostaglandins, thromboxanes, and leukotrienes. Of a range of sesquiterpene lactones of the germacrane and guianane groups characterized in the leaf material, the principal constituent and major active component is parthenolide (Figure 5.34) (up to about 1% in dried leaves). The powerful pungent odour of the plant arises from the volatile oil constituents, of which the monoterpene camphor (Figure 5.14) is a major constituent. Feverfew may be taken as the fresh leaf, often eaten with bread in the form of a sandwich to minimize the bitter taste, or it can be obtained in commercial dosage forms as tablets or capsules of the dried powdered leaf. The parthenolide content of dried leaf deteriorates on storage, and many commercial preparations of feverfew have been shown to contain little parthenolide, or to be well below the stated content. This may be a consequence of complexation with plant thiols via Michael addition. Consumers of fresh leaf can be troubled by sore mouth or mouth ulcers, caused by the sesquiterpenes. Parthenolide is also known to be capable of causing some allergic effects, e.g. contact dermatitis. The proposed mechanism of action of parthenolide via alkylation of thiol groups in proteins is shown in Figure 5.35.

– H+

O2 NADPH

E1

E2

NADP+ E3 CH2OH

germacryl cation

(+)-germacrene A

CHO

sequential oxidation of alcohol to acid E3 O2 NADPH

− H2O E4 O

O

O O

parthenolide

O (+)-costunolide

E1: (+)- germacrene A synthase (FPP is substrate for E1)

NADP+

E4 OH

lactone formation

E2: (+)-germacrene A hydroxylase E3: dehydrogenase

CO2H

CO2H

E4: (+)-costunolide synthase

Figure 5.34 transannular cyclization initiated via protonated epoxide; compare formation of guaiyl cation, Figure 5.31

O

O

R S H H

H

H OH O

H OH O

O H parthenolide

O H

Figure 5.35

attack of sulfhydryl in protein onto resultant carbocation

O

SR

nucleophilic attack of sulfhydryl onto α,β-unsaturated lactone: Michael reaction SR H R H S H OH O

O SR

H

HO H

O O

SR O

H

O

H

SR O sesquiterpene lactone irreversibly bound to protein

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 215 elimination of acetic acid and two molecules H2O; consider lactone as hydrolysed to hydroxyacid

OAc H

Δ – HOAc – 2 H2O

H OH O matricin

O

– CO2

HO2C chamazulene carboxylic acid

HO2C chamazulene

ibuprofen

Figure 5.36 α-Santonin (Figure 5.33) has been identified as the principal anthelmintic component of various Artemisia species, e.g. wormseed (A. cinia; Compositae/Asteraceae), and has found considerable use for removal of roundworms, although potential toxicity limits its application. Structurally, α-santonin bears much similarity to parthenolide, and the most marked difference lies in the presence of the bicyclic decalin ring system. This basic skeleton, the eudesmane system, is formed from the germacryl cation by protonation and cyclization via the eudesmyl cation (Figure 5.31, route ii), whereas protonation at the more substituted end of a double bond (anti-Markovnikov addition, route i), could generate the guaiyl cation and guaiane skeleton. This latter skeleton is found in matricin (Figure 5.36), again from matricaria flowers [Box 5.5]. This compound degrades on heating, via hydrolysis then elimination of acetic acid and water, followed by decarboxylation to the azulene derivative

chamazulene, which is responsible for the blue coloration of oil distilled from the flowers. Chamazulene carboxylic acid (Figure 5.36) is also found in yarrow (Achillea millefolium; Compositae/Asteraceae) and has been shown to be an anti-inflammatory agent by inhibiting cyclooxygenase COX-2, though not COX-1 (see page 62). The structural resemblance to the synthetic analgesic ibuprofen is striking. Thapsigargin (Figure 5.33) from seeds and roots of the Mediterranean plant Thapsia garganica (Compositae/Asteraceae) provides a further example of a guaianolide, highly oxygenated and esterified with a variety of acid groups. This compound is of considerable pharmacological interest in the study of Ca2+ signalling pathways. Thapsigargin is a potent inhibitor of Ca2+ -ATPases, and is capable of severely unbalancing cellular Ca2+ concentrations, often leading to disrupted cell function and growth, and apoptosis. A thapsigargin pro-drug has been tested in the treatment of prostate cancer.

Box 5.5 Chamomile and Matricaria Two types of chamomile (camomile) are commonly employed in herbal medicine: Roman chamomile Chamaemelum nobile (formerly Anthemis nobilis) (Compositae/Asteraceae) and German chamomile Matricaria chamomilla (Chamomilla recutica) (Compositae/Asteraceae). German chamomile, an annual plant, is the more important commercially and is often called matricaria to distinguish it from the perennial Roman chamomile. Both plants are cultivated in various European countries to produce the flower-heads which are then dried for drug use. Volatile oils obtained by steam distillation or solvent extraction are also available. Matricaria is also used as a digestive aid, but is mainly employed for its anti-inflammatory and spasmolytic properties. Extracts or the volatile oil find use in creams and ointments to treat inflammatory skin conditions, and as an antibacterial and antifungal agent. Taken internally, matricaria may help in the control of gastric ulcers. The flowers yield 0.5–1.5% volatile oil containing the sesquiterpenes α-bisabolol (10–25%), bisabolol oxides A and B (10–25%) (Figure 5.32), and chamazulene (0–15%) (Figure 5.36). Chamazulene is a thermal decomposition product from matricin, and is responsible for the dark blue coloration of the oil (Roman chamomile oil contains only trace amounts of chamazulene). α-Bisabolol has some anti-inflammatory, antibacterial, and ulcer-protective properties, but chamazulene probably contributes to the anti-inflammatory activity of matricaria preparations. It has been found to block the cyclooxygenase enzyme COX-2, though not COX-1, in prostaglandin biosynthesis (see page 62); the anti-inflammatory activity may result from the subsequent inhibition of leukotriene formation. Matricin itself produces rather greater anti-inflammatory activity than chamazulene and appears to be metabolized in the body to chamazulene carboxylic acid, a natural analogue of the synthetic analgesic ibuprofen (Figure 5.36).

216 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.5 (continued) Roman chamomile is usually taken as an aqueous infusion (chamomile tea) to aid digestion, curb flatulence, etc., but extracts also feature in mouthwashes, shampoos, and many pharmaceutical preparations. It has mild antiseptic and anti-inflammatory properties. The flower-heads yield 0.4–1.5% of volatile oil, which contains over 75% of aliphatic esters of angelic, tiglic, isovaleric, and isobutyric acids (Figure 5.33), products from metabolism of the amino acids isoleucine, leucine, and valine (see pages 56, 79, 316), with small amounts of monoterpenes and sesquiterpenes.

Another type of decalin-containing sesquiterpene is seen in the structures of cadinenes and amorpha4,11-diene. α-Cadinene (Figure 5.38) is one of the many terpenoids found in juniper berries (Juniperus communis; Cupressaceae) used in making gin, and this compound is derived from the 10-carbon ring-containing cis-germacryl cation. The double bonds in the cis-germacryl cation are unfavourably placed for a cyclization reaction, as observed with the germacryl cation, and available evidence points to an initial 1,3-shift of hydride to the

The 11-carbon ring of the humulyl carbocation (Figure 5.31) may be retained, as in the formation of α-humulene (Figure 5.37), or modified to give the caryophyllyl cation containing a nine-membered ring fused to a four-membered ring, as in β-caryophyllene (Figure 5.37). Humulene is found in hops (Humulus lupulus; Cannabaceae), and β-caryophyllene is found in a number of plants, e.g. in the oils from cloves (Syzygium aromaticum; Myrtaceae) and cinnamon (Cinnamomum zeylanicum; Lauraceae).

≡

– H+ a

b a H

E1

humulyl cation E2

α-humulene

b – H+

H E1: α-humulene synthase E2: β-caryophyllene synthase (FPP is substrate for both enzymes)

E2 H

β-caryophyllene

caryophyllyl cation

Figure 5.37

H − H+

H − H+

E1 H

H

H

δ-cadinene

cadinyl cation

α-cadinene

E1: δ-cadinene synthase (FPP or nerolidyl PP is substrate)

Figure 5.38

H

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 217 H

H

E1

H

amorphyl cation

E2

H

H

HO

amorpha-4,11-diene

H

H

H

O2 NADPH

E2

H

E2

H

H HO2C

OHC

artemisinic alcohol

artemisinic aldehyde

artemisinic acid reduction

O

hydrolysis of hemiketal HO

H

O H HO2C

HO

HOO O HO2C

formation of hemiacetal, then lactone

H

H HO2C

H

dihydroartemisinic acid

O

O O HO2C

O O

H

O HO

H

H HO2C

dihydroartemisinic acid hydroperoxide

hemiketal formation

H

O2/hν O

H HO2C

H

photooxidation to hydroperoxide

H

H2O

air oxidation to hydroperoxide

O2 O

H

O

H

cleavage of hydroperoxide, ring expansion, and quenching of carbocation

O H

E1: amorpha-4,11-diene synthase (FPP is substrate) E2: CYP sesquiterpene oxidase

O O

H

the conversion of dihydroartemisinic acid into artemisinin is thought to be a non-enzymic autooxidation

O artemisinin (qinghaosu)

Figure 5.39 isopropyl side-chain to generate a new cation, and thus allowing cyclization to the cadinyl cation (Figure 5.31). δ-Cadinene is an alternative deprotonation product; it is further elaborated to gossypol in cotton (see page 220). Amorpha-4,11-diene (Figure 5.39) is structurally related to the cadinenes, but the different stereochemistry of ring fusion and the position of the second double bond is a consequence of a different cyclization mechanism operating to produce the initial decalin ring system. In this case, a six-membered ring is formed first to give the bisabolyl cation; again, a 1,3-hydride shift is implicated prior to forming the decalin system of the amorphyl cation (Figure 5.31). Amorpha-4,11-diene is an intermediate in the pathway leading to artemisinin in Artemisia annua (Compositae/Asteraceae) (Figure 5.39). Artemisinic acid is formed from amorphadiene via modest oxidation processes all catalysed by a single cytochrome P-450-dependent enzyme system. Reduction to dihydroartemisinic acid is

followed by transformation into artemisinin by a sequence of reactions that is currently regarded as non-enzymic, and brought about by an oxygen-mediated photochemical oxidation under conditions that might normally be present in the plant. An intermediate in this process, also found naturally in A. annua, is the hydroperoxide of dihydroartemisinic acid. The further modifications postulated in Figure 5.39 include ring expansion by cleavage of this hydroperoxide and a second oxygen-mediated hydroperoxidation. The 1,2,4-trioxane system in artemisinin can be viewed more simply as a combination of hemiketal, hemiacetal, and lactone functions, and the later stages of the pathway merely reflect their construction. Artemisinin is an important antimalarial component in Artemisia annua, a Chinese herbal drug [Box 5.6]. There is currently strong research effort to produce artemisinin or analogues as new antimalarial drugs, since the malarial parasite has developed resistance to many of the current drugs (see quinine, page 382).

218 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.6 Artemisia annua and Artemisinin Artemisia annua (Compositae/Asteraceae) is known as qinghao in Chinese traditional medicine, where it has been used for centuries in the treatment of fevers and malaria. The plant is sometimes called annual or sweet wormwood, and is quite widespread, being found in Europe, North and South America, as well as China. Artemisinin (qinghaosu; Figure 5.40) was subsequently extracted and shown to be responsible for the antimalarial properties, being an effective blood schizontocide in humans infected with malaria, and showing virtually no toxicity. Malaria is caused by protozoa of the genus Plasmodium, especially P. falciparum, entering the blood system from the salivary glands of mosquitoes, and worldwide is responsible for 2–3 million deaths each year. Established antimalarial drugs, such as chloroquine (see page 382), are proving less effective in the treatment of malaria due to the appearance of drug-resistant strains of P. falciparum. Artemisinin is currently effective against these drug-resistant strains. Artemisinin is a sesquiterpene lactone containing a rare peroxide linkage which appears to be essential for activity. Some plants of A. annua have been found to produce as much as 1.4% artemisinin, but the yield is normally very much less, typically 0.05–0.2%. Apart from one or two low-yielding species, the compound has not been found in any other species of the genus Artemisia (about 400 species). A. annua is grown for drug use in China, Vietnam, East Africa, the United States, Russia, India, and Brazil. Small amounts (about 0.01%) of the related peroxide structure artemisitene (Figure 5.40) are also present in A. annua, though this has a lower antimalarial activity. The most abundant sesquiterpenes in the plant are artemisinic acid (arteannuic acid, qinghao acid; typically 0.2–0.8%) (Figure 5.39), and lesser amounts (0.1%) of arteannuin B (qinghaosu-II; Figure 5.40). Fortunately, the artemisinic acid content may be converted chemically into artemisinin by a relatively straightforward and efficient process. Artemisinin may also be reduced to the lactol (hemiacetal) dihydroartemisinin (Figure 5.40), and this has been used for the semi-synthesis of a range of analogues, of which the acetals artemether and arteether (Figure 5.40), and the water-soluble sodium salts of artelinic acid and artesunic acid (Figure 5.40) appear very promising antimalarial agents. These materials have increased activity compared with artemisinin, and the chances of infection recurring are also reduced. Artemether has rapid action against chloroquinine-resistant P. falciparum malaria, and is currently being used in combination with the

H

H O

O O

O H

O

O O

≡

H

O O

O H

O

O O

O artemisinin

O

H

O

O arteannuin B

O artemisitene H

H O O

O

H O

O

O H

O

H

O

H

O

O H

HO2C

O

O

O

H

O N

O S O O artemisone

artesunic acid

OH O O

O O

NH O

yingzhaosu A

Figure 5.40

O

O H

R = Me, artemether R = Et, arteether HO2C

OH

O

O

O H

artelinic acid

HO

O

H OR

dihydroartemisinin

H

H O

O

O O

H OH

O

yingzhaosu C

arterolane

NH2

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 219

Box 5.6 (continued) genes upregulated

genes downregulated

FPP

MVA

Artemisia annua gene encoding amorphadiene synthase

host organism: Saccharomyces cerevisiae

sterols Artemisia annua P-450 gene

H

H

H

H

semi-synthesis artemisinin

H

amorphadiene

HO

H

H

H

OHC

HO2C

artemisinic acid

Figure 5.41 synthetic antimalarial lumefantrine. Arteether has similar activity. Being acetals, artemether and arteether are both extensively decomposed in acidic conditions, but are stable in alkali. The ester artesunic acid (artesunate) is also used in injection form, but is rather unstable in alkaline solution, hydrolysing to dihydroartemisinin. The ether artelinic acid is considerably more stable. These two compounds have a rapid action and particular application in the treatment of potentially fatal cerebral malaria. Dihydroartemisinin is a more active antimalarial than artemisinin and appears to be the main metabolite of these drugs in the body; unfortunately, dihydroartemisinin displays some neurotoxicity. Nevertheless, these agents rapidly clear the blood of parasites, though they do not have a prophylactic effect. Artemisone (Figure 5.40), a new semi-synthetic thiomorpholine dioxide derivative of dihydroartemisinin, is currently in clinical trials. Chemically, these agents are quite unlike any other class of current antimalarial agent, are well tolerated with no major side-effects, and so far no drug resistance is evident. As new analogues are introduced, they may well provide an important group of drugs in the fight against this life-threatening disease. Currently, there is no shortage of Artemisia for drug use. However, as artemisinin derivatives become more widely used, the longer term supply of artemisinin for drug manufacture may need addressing. A significant development then is the use of genetic engineering to produce artemisinic acid in culture based on the biosynthetic pathway deduced for artemisinin (Figure 5.39). Genes from A. annua encoding the enzymes amorphadiene synthase and the cytochrome P-450-dependent monooxygenase that performs the three-step oxidation of amorphadiene to artemisinic acid were expressed in yeast Saccharomyces cerevisiae (Figure 5.41). To provide sufficient FPP precursor in the yeast host, several genes linked to the MVA → FPP transformation were upregulated; overexpression of the gene for HMGCoA reductase (see page 190) had the most significant effect. To avoid FPP being channelled off into steroid biosynthesis, the gene encoding for squalene synthase (see page 235) was downregulated. The net effect was high production of artemisinic acid (up to 100 mg l−1 ) in the culture medium. This breakthrough is being commercialized to provide artemisinic acid for semi-synthesis of antimalarial drugs. The relationship between a peroxide linkage and antimalarial activity is strengthened by the isolation of other sesquiterpene peroxides which have similar levels of activity as artemisinin. Thus, roots of the vine yingzhao (Artabotrys uncinatus; Annonaceae) which is also used as a traditional remedy for malaria, contain the bisabolyl derivatives yingzhaosu A and yingzhaosu C (Figure 5.40), the latter structure containing an aromatic ring of isoprenoid origin (compare the monoterpenes thymol and carvacrol, page 204). Arterolane (Figure 5.40) is a totally synthetic 1,2,4-trioxolane developed from the artemisinin template that shows potent antimalarial activity and is currently in clinical trials. Despite intensive studies, there is as yet no generally accepted mechanism of action for artemisinin and derivatives. The malarial parasite utilizes the host’s haemoglobin as a food source. However, haem, which is a soluble iron–porphyrin material released from haemoglobin as a result of proteolytic digestion, is toxic to Plasmodium, so it is normally converted into the insoluble non-toxic form haemozoin (malarial pigment) by enzymic polymerization. Agents like chloroquine (see page 382) may interfere with this polymerization process, or simply prevent the haemozoin from crystallizing. Of the various mechanisms proposed for the mode of action of artemisinin, initial reductive cleavage of the peroxide function by the FeII centre of haem to generate an oxygen radical species is widely accepted. This goes on to produce a carbon-centred radical that can interact with and effectively alkylate the porphyrin ring of haem. Alternatively, specific proteins or other biomolecules may suffer alkylation, resulting in death of the malarial parasite. It has also been suggested that artemisinin may act on a single enzymic target, a Ca2+ -ATPase; evidence for this comes from the observation that parasites with a mutant enzyme were insensitive to artemisinin.

220 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition various oxidative processes

OH

H

O2 NADPH E1

E2

H cadinyl cation

O OH

this is a cyclic ether of the primary alcohol OH

CH2OH

OH

H

H

(+)-δ-cadinene

8-hydroxy-δ-cadinene

desoxyhemigossypol O

O

HO

OH

CHO OH

one-electron oxidation

CHO

OH

CHO

radical coupling

OH

OH

H2O2

OH

×2

OH

OH

E3

OH

CHO OH

HO

O

CHO

resonance forms of radical

OH

gossypol

hemigossypol E1: (+)-δ-cadinene synthase E2: (+)-δ-cadinene 8-hydroxylase E3: peroxidase (FPP is substrate for E1)

Figure 5.42 Gossypol (Figure 5.42) is an interesting and unusual example of a dimeric sesquiterpene in which loss of hydrogen has led to an aromatic system (compare the phenolic monoterpenes thymol and carvacrol, page 204). The cadinyl carbocation via δ-cadinene is involved in generating the basic aromatic sesquiterpene unit hemigossypol, and then dimerization is simply an example of phenolic oxidative coupling ortho to the

phenol groups (Figure 5.42). The coupling is catalysed by an H2 O2 -dependent peroxidase enzyme. Gossypol is found in immature flower buds and seeds of the cotton plant (Gossypium species; Malvaceae), though it was originally isolated in small amounts from cottonseed oil. Its toxicity renders cottonseed oil unsafe for human consumption, but it has been used in China as a male infertility agent [Box 5.7].

Box 5.7 Gossypol Gossypol occurs in the seeds of cotton (Gossypium species, e.g. G. hirsutum, G. herbaceum, G. arboreum, G. barbadense; Malvaceae) in amounts of 0.1–0.6%. Cotton is a major plant crop, and a considerable amount of cottonseed is produced as a by-product; this is mainly used as a feedstuff for cattle. Cottonseed, though protein-rich, has limited food use because of the cardiotoxic and hepatotoxic effects of gossypol in humans and animals other than ruminants. The contraceptive effects of gossypol were discovered when subnormal fertility in some Chinese rural communities was traced back to the presence of gossypol in dietary cottonseed oil. Gossypol acts as a male contraceptive, altering sperm maturation, spermatozoid motility, and inactivation of sperm enzymes necessary for fertilization. Extensive clinical trials in China have shown the antifertility effect is reversible after stopping the treatment provided that consumption has not been too prolonged. Cases of irreversible infertility have resulted from longer periods of drug use. The gossypol molecule is chiral due to restricted rotation about the aryl–aryl linkage, and can thus exist as two atropisomers which do not easily racemize (Figure 5.43). Only the (−)-isomer is pharmacologically active as a contraceptive, whereas most of the toxic symptoms appear to be associated with the (+)-isomer. Most species of Gossypium (except G. barbadense)

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 221

Box 5.7 (continued) OHC HO

OH

HO OH

HO

CHO OH

HO

OH OH HO

HO

CHO

OHC (+)-gossypol

OH

(–)-gossypol

Figure 5.43

produce gossypol where the (+)-isomer predominates over the (−)-isomer, with amounts varying according to species and cultivar. The relative proportions of the two isomers appear to be controlled by regioselective coupling of two hemigossypol molecules by the peroxidase enzyme (Figure 5.42). Racemic (±)-gossypol (but neither of the enantiomers) complexes with acetic acid, so that suitable treatment of cottonseed extracts can separate the racemate from the excess of (+)-isomer. The racemic form can then be resolved. Other plants in the Gossypieae tribe of the Malvaceae also produce gossypol, with the barks of Thespia populnea (3.3%) and Montezuma speciosissima (6.1%) being particularly rich sources. Unfortunately, gossypol from these sources is almost entirely the (+)-form that lacks contraceptive activity. An exception is Thespia danis, where aerial parts contain predominantly (72%) (−)-gossypol; yields are about 0.2%. It is possible to reduce gossypol levels in cottonseed by interfering with the enzyme δ-cadinene synthase in the plant; this has been achieved by expression of the antisense gene, or by gene silencing through RNA interference: supplying a short strand of RNA to target that portion of mRNA responsible for a particular enzyme. Such techniques may lead to production of strains yielding non-toxic cottonseed oil.

The formation of sesquiterpenes by a carbocation mechanism also means that there is considerable scope for rearrangements of the Wagner–Meerwein type. So far, only occasional hydride migrations have been invoked in rationalizing the examples considered. Obviously, fundamental skeletal rearrangements will broaden the range of natural sesquiterpenes even further. That such processes do occur has been proven beyond doubt by appropriate labelling experiments, and a single example will be used as illustration. The trichothecenes are a group of fungal toxins found typically in infected grain foodstuffs [Box 5.8]. Their name comes from the fungal genus Trichothecium, but most of the known structures are derived from cultures of Fusarium species. A typical trichothecene contaminant is 3-acetyldeoxynivalenol, which is produced from the less-substituted trichothecene isotrichodermol by a sequence of oxygenation, esterification, and de-esterification reactions (Figure 5.44). The trichothecenes have their origins in nerolidyl diphosphate, and ring closure of the bisabolyl cation derived from it generates a new carbocation with a five-membered ring (Figure 5.44). At this stage, a series of one hydride and two methyl migrations occur to give a cation, which loses a proton to produce the specific trichothecene

precursor trichodiene. These migrations are fully backed up by experimental data and, although not immediately predictable, can be rationalized satisfactorily by consideration of the cation suitably bound to the enzyme surface, as shown in Figure 5.44. The sequence is initiated by a 1,4-hydride shift, which is spatially allowed by the relative proximity of the centres. Two 1,2-methyl shifts then follow, and it is important to note that each migrating group attacks the opposite side of the centre from which the previous group is departing, i.e. inverting the configuration at these centres. Accordingly, a concerted sequence of migrations is feasible; a more vivid example of this type of concerted process is seen in the formation of triterpenoids and steroids (see page 236). Loss of a proton and generation of a double bond then terminates the process, giving trichodiene. Oxygenation of trichodiene gives, in several steps, isotrichotriol. Two of the hydroxylations are at activated allylic positions; hydroxylation on the five-membered ring, therefore, will occur before the epoxidation. Ether formation, involving perhaps protonation, loss of water, and generation of an allylic cation completes the pathway to the basic trichothecene structure as in isotrichodermol.

222 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition electrophilic addition leads to five-membered ring and secondary cation H

11

H E1

E1

H

≡

bisabolyl cation Wagner–Meerwein rearrangements: 1,4-hydride shift; two 1,2-methyl shifts

≡

2

3

trichodiene

H H

2α-hydroxylation

O2, NADPH E2

epoxidation 11α-hydroxylation 3α-hydroxylation

nucleophilic attack of hydroxyl onto allylic cation H

O

H

H

OAc acetylCoA

O

H

O

OH

OH O

HO OH

+ OH + H – H 2O

O

OH O

E3 isotrichodermin O2, NADPH

E4

H

H

O

isotrichodermol

H

OAc acetylCoA O

O

allylic cation 7α-hydroxylation; 8α-hydroxylation; OAc 15-deacetylation; oxidation of 8-OH

H

HO 15-deacetylcalonectrin E1: trichodiene synthase (FPP is substrate) E2: TRI4 (trichodiene 2α-hydroxylase)

10

9 8

O

calonectrin

O

15

H

13 6

7

HO

AcO

H 11

O E5

isotrichotriol

OAc

3

2

O 12

5

4

14

OH 3-acetyldeoxynivalenol

E3: TRI101 (isotrichodermol 3-O-acetyltransferase) E4: TRI11 (isotrichodermin 15-hydroxylase) E5: TRI3 (15-deacetylcalonectrin 15-O-acetyltransferase)

Figure 5.44

Box 5.8 Trichothecenes The trichothecenes are a group of sesquiterpene toxins produced by several fungi of the genera Fusarium, Myrothecium, Trichothecium, and Trichoderma, which are parasitic on cereals such as maize, wheat, rye, barley, and rice. About 180 different structures have been identified, with some of these being isolated from plants of the genus Baccharis (Compositae/Asteraceae), where a symbiotic plant–fungus relationship may account for their production. Examples of trichothecene structures most commonly encountered as food contaminants include deoxynivalenol (DON; vomitoxin), diacetoxyscirpenol, T-2 toxin, and verrucarin A (Figure 5.45). The double bond and the epoxide group in the basic trichothecene skeleton are essential for toxicity, and the number of oxygen substituents and ester functions also contribute. Macrocyclic ester functions as seen in verrucarin A tend to produce the most toxic examples. Although these compounds are more toxic when injected, oral toxicity is relatively high, and lethal amounts can easily be consumed because of the nature of the host plants. They are sufficiently toxic to warrant routine analysis of foodstuffs such as wheat and flour, and also flour-derived products, e.g. bread, since they survive processing and the high temperatures used in baking. DON levels above 1 ppm are considered hazardous for human consumption. It is relevant to note that, when mammals ingest these compounds, a degree of de-epoxidation can occur, ascribed to gut microflora, thus providing a level of detoxification by removing a structural feature necessary for toxicity. As their main mechanism of action, these compounds inhibit protein biosynthesis by binding to the ribosome and inhibiting peptidyl transferase activity (see page 422). They also inhibit DNA biosynthesis. A major human condition known to be caused by

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 223

Box 5.8 (continued) H H

O

H

H

OH O

O

H

H

OH O

O

O

O

H

OH deoxynivalenol (DON)

OAc

AcO

H

O

O O OAc

AcO

HO diacetoxyscirpenol (DAS)

O

O

OH

O HO

O

O O

T-2 toxin

O verrucarin A

Figure 5.45 trichothecenes is alimentary toxic aleukia (ATA), characterized by destruction of the skin, haemorrhaging, inflammation, sepsis, a decrease in red and white blood corpuscles, bone marrow atrophy, and a high mortality rate. A severe outbreak of ATA was recorded in the former Soviet Union shortly after the Second World War when food shortages led to the consumption of grain that had overwintered in the field. This had become badly contaminated with Fusarium sporotrichioides and, hence, T-2 toxin. It is estimated that tens of thousands died as a result. Many trichothecene derivatives have been tested as potential anticancer agents, but they have proved too toxic for clinical use.

Finally, it is worth noting how many of the sesquiterpene derivatives described above are found in plants belonging to the daisy family, the Compositae/Asteraceae. Whilst sesquiterpenes are by no means restricted to this family, the Compositae/Asteraceae undoubtedly provides a very rich source.

DITERPENES (C20 ) The diterpenes arise from geranylgeranyl diphosphate (GGPP), which is formed by addition of a further IPP molecule to FPP in the same manner as described for the lower terpenoids (Figure 5.46). One of the

OPP farnesyl PP (FPP) E1

OPP IPP

allylic cation

electrophilic addition gives tertiary cation

OPP HR HS E1

stereospecific loss of proton

OPP geranylgeranyl PP (GGPP) E1: geranylgeranyl diphosphate synthase

Figure 5.46

224 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

OH phytol

O

N

N Mg2+ N

N O phylloquinone (vitamin K1)

MeO2C

HO O

O

O

O chlorophyll a

α-tocopherol (vitamin E)

Figure 5.47 simplest and most important of the diterpenes is phytol (Figure 5.47), a reduced form of geranylgeraniol, which forms the lipophilic side-chain of the chlorophylls, e.g. chlorophyll a (Figure 5.47). Related haem molecules, the porphyrin components of haemoglobin, lack such lipophilic side-chains. Available evidence suggests that GGPP is involved in forming the ester linkage, and the three reduction steps necessary to form the phytol ester occur after attachment to the chlorophyll molecule. A phytyl substituent is also found in vitamin K1 (phylloquinone; Figure 5.47), a naphthoquinone derivative found in plants, though other members of the vitamin K group (menaquinones) from bacteria have unsaturated terpenoid side-chains of variable length. The phytyl group of phylloquinone is introduced by alkylation of dihydroxynaphthoic acid with phytyl diphosphate and a similar phytylation of homogentisic acid features in the formation of the E group vitamins (tocopherols). These compounds are discussed further under shikimate derivatives (see page 178). Cyclization reactions of GGPP mediated by carbocation formation, plus the potential for Wagner–Meerwein rearrangements, will allow many structural variants of

diterpenoids to be produced. The toxic principle ‘taxine’ from English yew (Taxus baccata; Taxaceae) has been shown to be a mixture of at least 11 compounds based on the taxadiene skeleton, which can readily be rationalized as in Figure 5.48, employing the same mechanistic principles as seen with mono- and sesqui-terpenes. A novel feature discovered is the enzyme’s ability to utilize the proton released during alkene formation to protonate another double bond; this effectively relocates the cationic centre. Although these compounds are sometimes classified as diterpenoid alkaloids, the nitrogen atom is not incorporated into the diterpene skeleton, as exemplified by taxol (paclitaxel; Figure 5.48) from Pacific yew (Taxus brevifolia) [Box 5.9]. The side-chains in taxol containing aromatic rings are derived from shikimate via phenylalanine. The nitrogen atom derives from the amino acid β-phenylalanine, a rearranged version of the α-amino acid l-phenylalanine. Virtually all of the enzymes and genes for the whole sequence from GGPP to taxol are now characterized. Some enzymes show relatively broad substrate specificity. Taxol is an important anticancer agent, with a broad spectrum of activity against some cancers that do not respond to other agents.

Box 5.9 Taxus brevifolia and Taxol (Paclitaxel) A note on nomenclature: the name taxol was given to a diterpene ester with anticancer properties when it was first isolated in 1971 from Taxus brevifolia. However, the name Taxol had already been registered as a trademark; this was subsequently used when the natural product was exploited commercially as a drug. Accordingly, the generic name paclitaxel has been assigned to the

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 225

cyclization by electrophilic addition giving tertiary cation

electrophilic addition giving tertiary cation

E

intramolecular transfer of proton; proton lost in alkene formation is used to protonate another double bond and give tertiary cation

H

E E1

E OPP GGPP

verticillyl cation OH

O

10

HO

11

12

9 16

E5

β-phenylalanoyl-CoA

E6

O2, NADPH

E7

benzoyl-CoA

E8

H

acetylO2 CoA O2 NADPH NADPH

19 8 7 6 3 4 5

E4

E3

– H+

E2

E1

5

OAc

further esterifications plus side-chain hydroxylation

O O 2′

O

O O

O O

O

O O

Ph

OH

NH

O

Ph

O

H O AcO

CO2H NH2

Figure 5.48

H

E6: E7: E8: E9:

NH2 CO2H

E9

L-Phe taxadiene synthase taxoid 5α-hydroxylase taxadienol O-acetyltransferase taxoid 10β-hydroxylase 10β-O-acetyltransferase

O

paclitaxel (taxol)

paclitaxel (taxol)

E1: E2: E3: E4: E5:

O

OH O

phenylalanine aminomutase reaction:

O

AcO

H

O

OH

CH3

O

HO

Ph

H

OH HO O

taxenyl cation

postulated oxetane ring formation:

AcO

NH

H

taxadiene

O

O

H

H

OAc 5 a-hydroxylation 20 via allylic radical; 5 a-acetylation; taxadien10b-hydroxylation 5α-acetoxy-10β-ol

10-deacetylbaccatin III acetyl-CoA

17

1 2

several steps; H O hydroxylations and esterifications

O AcO

10

13 15 14

H HO O

OH

18

OH

electrophilic cyclization

baccatin III 13-O-phenylpropanoyltransferase β-phenylalanoylbaccatin III 2′-hydroxylase taxane N-benzoyltransferase phenylalanine aminomutase

(3R)-β-phenylalanine

226 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.9 (continued) compound, and the literature now contains an unhappy mixture of the two names, though the original name taxol is most often employed. The anticancer drug taxol (Figure 5.48) is extracted from the bark of the Pacific yew, T. brevifolia (Taxaceae), a slow-growing shrub/tree found in the forests of northwest Canada (British Columbia) and the USA (Washington, Oregon, Montana, Idaho, and north California). Although the plant is not rare, it does not form thick populations, and it needs to be mature (about 100 years old) to be large enough for exploitation of its bark. The bark from about three mature trees is required to provide 1 g of taxol, and a course of treatment may need 2 g of the drug. Current demand for taxol is in the region of 250 kg per annum. Harvesting has been strictly regulated, but it was immediately realized that this source would not provide a satisfactory long-term supply of the drug. Taxol is now obtained by alternative means. All parts of T. brevifolia contain a wide range of diterpenoid derivatives termed taxanes, which are structurally related to the toxic constituents found in other Taxus species, e.g. the common English yew, T. baccata. Nearly 400 taxanes have been characterized from various Taxus species, and taxol is a member of a small group of compounds possessing a four-membered oxetane ring and a complex ester side-chain in their structures; both of these features are essential for antitumour activity. Taxol is found predominantly in the bark of T. brevifolia, but in relatively low amounts (about 0.01–0.02%). Up to 0.033% of taxol has been recorded in some samples of leaves and twigs, but generally the taxol content is much lower than in the bark. The content of some other taxane derivatives in the bark is considerably higher, e.g. up to 0.2% baccatin III (Figure 5.49). Other taxane derivatives characterized include 10-deacetyltaxol, 10-deacetylbaccatin III, cephalomannine, and 10-deacetylcephalomannine (Figure 5.49).

OR

O

10

HO

O

OH

NH

O

O

10

O

H

OH

AcO

O

PhCO2

R = Ac, baccatin III R = H, 10-deacetylbaccatin III

O

NH

O

PhCO2

NH

O O

OH

H

OR

AcO

larotaxel O

O R=

HO PhCO2

AcO

O

OH NH

O O

DHA-paclitaxel (taxoprexin)

14

OH

HO

O

AcO

R = H, docetaxel (taxotere) R = Me, cabazitaxel

O

H

O O

PhCO2

7

8

OH

7

H

O O

OR

HO

O

AcO

OAc

O

Figure 5.49

OH

R = Ac, cephalomannine R = H, 10-deacetylcephalomannine O

O

10

O O

HO

PhCO2

OR NH

O

H

OH

HO

O

O

OR

O

O

OH

H O

O ortataxel

O

OAc OCOPh

O

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 227

Box 5.9 (continued) A satisfactory solution currently exploited for the supply of taxol and derivatives for drug use is to produce these compounds by semi-synthesis from more accessible structurally related materials. Both baccatin III and 10-deacetylbaccatin III (Figure 5.49) have been efficiently transformed into taxol, the latter being the preferred substrate. 10-Deacetylbaccatin III is readily extracted from the leaves and twigs of English yew T. baccata, and although the content is variable, it is generally present at much higher levels (up to 0.2%) than taxol can be found in T. brevifolia. T. baccata is widely planted as an ornamental tree in Europe and the USA and is much faster growing than the Pacific yew. Yew trees, typically 8–10 years old, can be harvested by pruning off top growth on a regular basis. In Europe, there are also arrangements whereby unwanted clippings from ornamental yew hedges may be channelled into drug production. Semi-synthesis provided reliable supplies to establish taxol as an important anticancer drug. However, the chemical conversion of 10-deacetylbaccatin III is still complex enough to consider alternative approaches. Taxol-producing cell cultures of Chinese yew T . chinensis have been established and are currently grown in fermentors for commercial drug production. Taxol is isolated and purified from the fermentation broth; taxol yields in the region of 20 mg l−1 can be obtained from cultures of various Taxus species. Initial optimism for obtaining taxol by microbial culture was tempered by the very low levels produced. Fungi such as Taxomyces adreanae isolated from the inner bark of T . brevifolia, and Pestalotiopsis microspora from the inner bark of the Himalayan yew (T . wallachiana), appear to have inherited the ability to synthesize taxol by gene transfer from the host tree. At best, taxol levels measured were only about 70 μg l−1 of culture medium, and thus commercially insignificant. However, the use of microorganisms and enzymes to specifically hydrolyse ester groups from mixtures of structurally related plant-derived taxanes in crude extracts and thus improve the yields of 10-deacetylbaccatin III has been reported. Paclitaxel (Taxol®) is used clinically in the treatment of ovarian and breast cancers, non-small-cell lung cancer, small-cell lung cancer, and cancers of the head and neck. A disadvantage associated with taxol is its very low water solubility. Docetaxel (Taxotere®; Figure 5.49) is a side-chain analogue of taxol; it was one of the intermediates produced during a semi-synthesis of taxol from 10-deacetylbaccatin III. It was found to be more active than taxol, and, crucially, was more water-soluble. It is used against ovarian and breast cancers and non-small-cell lung cancer. Clinical development of taxol did not proceed until it was realized that it possessed a mode of action different from the drugs available at the time. Taxol acts as an antimitotic by binding to microtubules, promoting their assembly from tubulin, and stabilizing them against depolymerization during cell division. The resultant abnormal tubulin–microtubule equilibrium disrupts the normal mitotic spindle apparatus and blocks cell proliferation. Vincristine and vinblastine (see page 375), and podophyllotoxin (see page 155), also interfere with the tubulin system, but bind to the protein tubulin in the mitotic spindle, preventing polymerization and assembly into microtubules. Taxol thus shares a similar target, but has a different mechanism of action to the other antimitotics. In recent years, other natural products have been discovered that have the same or a similar mode of action; for example, the epothilones (see page 85) and discodermolide (see page 90). Taxol has also been shown to bind to a second target, a protein which normally blocks the process of apoptosis (cell death). Inhibition of this protein allows apoptosis to proceed. Several taxol analogues are in clinical trials. Considerable effort is being directed to improve solubility and to counter a tendency to induce multiple drug resistance. Four compounds in advanced clinical trials are DHA–paclitaxel (Taxoprexin®), cabazitaxel, larotaxel, and ortataxel (Figure 5.49). The first is an ester of taxol at the side-chain hydroxyl with the polyunsaturated fatty acid DHA (see page 49); polyunsaturated fatty acids are able to deliver cytotoxic drugs selectively to cancer cells. The other three structures are based on taxotere. Cabazitaxel is the 7,10-dimethyl ether and larotaxel is a 7,8-cyclopropane analogue. Ortataxel is an orally active cyclic carbonate derivative produced from the natural 14β-hydroxy-10-deacetylbaccatin III.

The latex of some plants in the genus Euphorbia (Euphorbiaceae) can cause poisoning in humans and animals, skin dermatitis, cell proliferation, and tumour promotion (co-carcinogen activity). Many species of Euphorbia are regarded as potentially toxic, and the latex can produce severe irritant effects, especially on mucous membranes and the eye. Most of the biological effects are due to diterpene esters, e.g. esters of phorbol (Figure 5.50), which activate protein kinase C, an important and widely distributed enzyme responsible for phosphorylating many biochemical entities. The permanent activation of protein kinase C is thought to lead to

the uncontrolled cancerous growth. The most commonly encountered ester of phorbol is 12-O-myristoylphorbol 13-acetate (Figure 5.50), one of the most potent tumour promoters known. The origins of phorbol are not known, but may be rationalized as in Figure 5.50. Cyclization of GGPP generates a cation containing a 14-membered ring system. Loss of a proton via cyclopropane ring formation leads to casbene, an antifungal metabolite produced by the castor oil plant, Ricinus communis (Euphorbiaceae). Casbene, via the ring closures shown in Figure 5.50, is then likely to be the precursor of the phorbol ring system.

228 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition electrophilic addition gives tertiary cation

formation of cyclopropane ring with loss of proton

OPP

H

H E1

E1 H

GGPP

casbene O CH3(CH2)12

O

HO

OAc esterifications

H

O

OH

H

H OH

proposed ring closures

H

OH

OH

O

HO 12-O-myristoylphorbol 13-acetate

O

H

OH

H various oxygenations

HO phorbol

E1: casbene synthase

Figure 5.50 In contrast to the cyclization mechanisms shown in Figures 5.48 and 5.50, where loss of diphosphate generates the initial carbocation, many of the natural diterpenes have arisen by a different mechanism. Carbocation formation is initiated by protonation of the double bond at the head of the chain, leading to a first cyclization sequence. Loss of the diphosphate later on also produces a carbocation and facilitates further cyclization. The early part of the sequence resembles that involved in hopanoid

biosynthesis (see page 242), and to some extent triterpenoid and steroid biosynthesis (see page 238), though in the latter cases it is the opening of the epoxide ring in the precursor squalene oxide rather than protonation of an alkene that is responsible for generation of the cationic intermediates. Protonation of GGPP can initiate a concerted cyclization sequence, terminated by loss of a proton from a methyl, yielding (−)-copalyl PP (Figure 5.51, a). The stereochemistry in this product is controlled by the folding

protonation of double bond gives tertiary carbocation; this allows a series of concerted cyclizations terminating in proton loss H

H

H

H

H

a OPP

E1

OPP

OPP

H

OPP

OPP

≡

b E2 H

H

GGPP E1: (−)-copalyl diphosphate synthase (ent-kaurene synthase A) E2: (+)-copalyl diphosphate synthase (part of bifunctional abietadiene synthase)

Figure 5.51

H (–)-copalyl PP

H H

≡

H

GGPP

H

OPP H

H H (+)-copalyl PP (labdadienyl PP)

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 229 cyclization of alkene onto cation produces secondary cation

loss of diphosphate generates allylic action; further cyclization of alkene onto cation

∇

OPP H

*

H

H E1

H

H

H ent-pimarenyl cation

(−)-copalyl PP O

sequential oxidation of methyl to carboxyl

H

ent-beyeranyl cation

H

O2 NADPH

E3

13

OH

H

∇

E1 H

H ent-kauranyl cation

ent-kaurene

ent-kaurenoic acid

*

H

E2

H CO2H

W–M 1,2-alkyl shift

loss of proton generates alkene

O2 NADPH

secondary cation converted into tertiary cation by 1,2-alkyl migration

O β2→1

glucosylations via 13-monoglucoside and 13-diglucoside

OGlc

3 × UDPGlc

CO2H

H

H E4

Glc

E5 E6

H CO2H steviol E1: kaurene synthase (ent-kaurene synthase B) E2: ent-kaurene oxidase

H CO2Glc stevioside

steviol

OH

E3: ent-kaurenoic acid 13-hydroxylase E4, E5, E6: glucosyltransferases

Figure 5.52 of the substrate on the enzyme surface, though an alternative folding can lead to (+)-copalyl PP (labdadienyl PP), the enantiomeric product with opposite configurations at the newly generated chiral centres (Figure 5.51, b). From (−)-copalyl PP, a sequence of cyclizations and a rearrangement, all catalysed by a single enzyme kaurene synthase, leads to ent-kaurene (Figure 5.52). As shown, this involves loss of the diphosphate leaving group enabling carbocation-mediated formation of the third ring system, and subsequent production of the fourth ring. Then follows a Wagner–Meerwein migration, effectively contracting the original six-membered ring to a five-membered one, whilst expanding the five-membered ring to give a six-membered ring. The driving force is transformation of a secondary carbocation to give a tertiary one, but this also results in the methyl group no longer being at a bridgehead, and what appears at first glance to be merely a confusing change in

stereochemistry. Loss of a proton from this methyl generates the exocyclic double bond of ent-kaurene and provides an exit from the carbocationic system. The prefix ent is used to indicate enantiomeric; the most common stereochemistry is that found in (+)-copalyl PP (Figure 5.51) and derivatives, so the kaurene series is termed ‘enantiomeric’. ent-Kaurene is the precursor of stevioside (Figure 5.52) in the plant Stevia rebaudiana (Compositae/Asteraceae) by relatively simple oxidation, hydroxylation, and glucosylation reactions. Both glucosyl ester and glucoside linkages are present in stevioside, and these help to confer an intensely sweet taste to this and related compounds. Stevioside is present in the plant leaf in quite large amounts (3–10%), is some 200–300 times as sweet as sucrose, and is being used in many countries as a commercial non-calorific sweetening agent.

230 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition GGPP E1

intramolecular transfer of proton; proton lost in alkene formation is used to protonate another double bond and give secondary cation

loss of diphosphate generates allylic action; further cyclization of alkene onto cation OPP

H

H

H

H

methyl migration to generate tertiary carbocation

E1 H

H

H sandaracopimarenyl cation

(+)-copalyl PP

W−M 1,2-methyl shift

sequential oxidation of methyl to carboxylic acid via alcohol and aldehyde

H

O2 NADPH H E2

E2

H

E1

H CO2H (–)-abietic acid

loss of proton generates diene

H O

H

(–)-abietadiene

H

abietenyl cation

E1: abietadiene synthase (bifunctional enzyme) E2: abietadienol/al oxidase

Figure 5.53 The alternative stereochemistry typified by (+)copalyl PP can be seen in the structure of abietic acid (Figure 5.53), the major component of the rosin fraction of turpentine from pines and other conifers (Table 5.1). Initially, the tricyclic system is built up as in the pathway to ent-kaurene (Figure 5.52), via the same mechanism, but generating the enantiomeric series of compounds. An internal proton transfer (compare taxadiene page 225) relocates the carbocationic centre to the side-chain, which then undergoes a methyl migration (Figure 5.53). Finally, loss of a proton leads to the diene abietadiene. The whole sequence from GGPP to abietadiene is catalysed by a single bifunctional enzyme; the first activity is a (+)-copalyl PP synthase, and the product is then transferred to the second active site. In the first step, carbocation formation is proton initiated, whilst the second is ionization initiated. Abietic acid results from sequential oxidation of the 4α-methyl. Wounding of pine trees leads to an accumulation at the wound site of both monoterpenes and diterpenes, and fractionation by distillation gives turpentine oil and rosin. The volatile monoterpenes seem to act as a solvent to allow deposition of the rosin layer to seal the wound. The diterpenes in rosin have both antifungal and insecticidal properties.

Extensive modification of the copalyl diterpene skeleton is responsible for generation of the ginkgolides, highly oxidized diterpene trilactones which are the active principles of Ginkgo biloba (Ginkgoaceae) [Box 5.10]. Apart from the C20 skeleton, hardly any typical terpenoid features are recognizable. However, a speculative scheme involving several rearrangements, ring cleavage, and formation of lactone rings can broadly explain its origin and provide a link between these extremely complex ginkgolide structures and the more familiar diterpenes (Figure 5.54). Detailed evidence is lacking. What is known is that levopimaradiene and abietatriene are precursors; levopimaradiene synthase is also a bifunctional enzyme (compare abietadiene synthase) that converts GGPP into levopimaradiene via (+)-copalyl PP. The unusual tert-butyl substituent arises as a consequence of the A ring cleavage. Bilobalide (Figure 5.54) contains a related C15 -skeleton, and is most likely a partially degraded ginkgolide. Ginkgo is the world’s oldest tree species, and its leaves are a highly popular and currently fashionable health supplement, taken in the anticipation that it can delay some of the degeneration of the faculties normally experienced in old age.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 231 GGPP E1 see Figure 5.53 OPP

– H+

H

O H

H E1

H

H

H abietenyl cation

(+)-copalyl PP

H

levopimaradiene

abietatriene (dehydroabietane)

E1: levopimaradiene synthase A ring cleavage

ginkgolides A

B

W–M 1,2-alkyl shift

further sequence of oxidative reactions ring closures as indicated (1 hemiacetal, 3 lactones)

OH

CO2H

O

O

W–M 1,2-alkyl shift

ginkgolides are usually drawn as if viewed O from the other side

O O

O

O

OHC HO2C

OH CO2H OH

O

O

O O ginkgolide

≡

O

O

O

O

O O

O loss of carbons as indicated; lactone formation from residual carboxyl and alcohol functions

O

O

e.g. bilobalide

Figure 5.54

Box 5.10 Ginkgo biloba Ginkgo biloba is a primitive member of the gymnosperms and the only survivor of the Ginkgoaceae, all other species being found only as fossils. It is a small tree native to China, but widely planted as an ornamental, and cultivated for drug use in Korea, France, and the United States. Standardized extracts of the leaves are marketed against cerebral vascular disease and senile dementia. Extracts have been shown to improve peripheral and cerebrovascular circulation. The decline in cognitive function and memory processes in old age can be due to disturbances in brain blood circulation, and thus ginkgo may exert beneficial effects by improving this circulation, and assist with other symptoms such as vertigo, tinnitus, and hearing loss. Virtually all clinical studies report positive results regarding cerebral insufficiency. The active constituents have been characterized as mixtures of terpenoids and flavonoids. The dried leaves contain 0.1–0.25% terpene lactones, comprising predominantly the five ginkgolides (A, B, C, J, and M) and bilobalide (Figure 5.55). Bilobalide accounts for about 30–40% of the mixture, whilst ginkgolide A is the predominant ginkgolide (about 30%). The ginkgolides are diterpenoid in nature, whilst bilobalide is described as sesquiterpenoid. However, bilobalide bears such a structural similarity to the ginkgolides, that it is most probably a degraded ginkgolide. The ginkgolides have been shown to have potent and selective antagonistic activity towards platelet-activating factor (PAF, see page 44), which is implicated in many physiological processes. More recently, ginkgolides have been shown to extert antagonistic effects on glycine receptors; together with GABA receptors, glycine receptors are the main inhibitory receptors in the central nervous system. Bilobalide lacks PAF antagonistic activity but shows neuroprotective effects. The flavonoid content of the dried leaves is 0.5–1.0%, and consists of a mixture of mono-, di-, and tri-glycosides of the flavonols kaempferol and quercetin (Figure 5.55; see also page 171) and some biflavonoids. These probably

232 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.10 (continued)

O

H

O O

HO R2

O

O R3

R1 O

OH

≡

O

O

OH OH

O

O

O

O

R1

R2

R3

ginkgolide A

OH

H

H

ginkgolide B

OH

OH

H

ginkgolide C

OH

OH

OH

ginkgolide J

OH

H

OH

ginkgolide M

H

OH

OH

R

O

O O

O

O

O

HO

R=

H

O bilobalide

OH HO

O

R OH

OH O R = H, kaempferol R = OH, quercetin 13:0 15:1 (Δ8)

CO2H OH ginkgolic acids

15:1 (Δ10) 17:1 (Δ8) 17:1 (Δ12)

Figure 5.55 also contribute to the activity of ginkgo, and may act as radical scavengers. Ginkgo leaf is also a rich source of shikimic and quinic acids (see page 138). Extracts of ginkgo for drug use are usually standardized to contain flavonoid glycosides and terpene lactones in a ratio of 24% to 6%, or 27% to 7%. Standardized extracts should also contain less than 5 ppm of alkyl phenols, since these are known to be allergenic, capable of inducing contact dermatitis, as well as being potentially cytotoxic and mutagenic. The ginkgolic acids (also known as anacardic acids, see page 118) form the main group of alkyl phenols found in ginkgo leaf (up to 1.7%). These phenolic acids have long, mainly unsaturated, alkyl chains and are related to the fatty acids (Figure 5.55). Ginkgo may be combined with ginseng (see page 245) in the treatment of geriatric disorders. Ginkgo and the ginkgolides are undergoing extensive investigation in conditions where there are high PAF levels, e.g. shock, burns, ulceration, and inflammatory skin disease.

In forskolin (colforsin; Figure 5.56), the third ring is heterocyclic rather than carbocyclic. The basic skeleton of forskolin can be viewed as the result of quenching of the cation by water as opposed to proton loss, followed by SN 2 -type nucleophilic substitution onto the allylic diphosphate (or nucleophilic substitution onto the allylic cation generated by loss of diphosphate) (Figure 5.56). A series of oxidative modifications will

then lead to forskolin. This compound has been isolated from roots of Coleus forskohlii (Plectranthus barbatus) (Labiatae/Lamiaceae), a plant used in Indian traditional medicine, and has been found to lower blood pressure and have cardioprotective properties. Forskolin has become a valuable pharmacological tool as a potent stimulator of adenylate cyclase activity, and has shown promising potential for the treatment of glaucoma,

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 233 loss of diphosphate and formation of allylic cation; quenching by attack of hydroxyl gives heterocyclic ring

quenching of intermediate cation by H2O OPP

OPP H

OH2 H

O

OH

H

H

H sequence of oxidation and esterification reactions

GGPP HO

O O

HO

O

O

OH H

OH

OAc O

colforsin daropate

H NMe2

O

OAc OH

forskolin (colforsin)

Figure 5.56

O

sequence of concerted W−M 1,2-hydride and 1,2-methyl shifts

GGPP

H

OPP

OPP

OPP

H

OPP

O

H

H

AcO

H

H

O O

H

H

H MeO2C labdanyl cation H

H

neoclerodiene PP

W−M shifts with inversion of stereochemistry

H

salvinorin A

H OPP

OPP labdanyl cation

Figure 5.57 congestive heart failure, hypertension, and bronchial asthma. The water-soluble derivative colforsin daropate has been introduced for drug use. In Figure 5.56, the GGPP-derived cation is quenched by the addition of water; in other cases, it may precipitate a series of concerted Wagner–Meerwein hydride and methyl migrations, resulting in a rearranged skeleton with different stereochemistry (Figure 5.57). Similar processes are encountered in triterpenoid

and steroid biosynthesis (see page 238). The first migration is of hydride, generating a new tertiary carbocation, with successive methyl, hydride, and methyl migrations; the sequence is terminated by proton loss, giving neoclerodiene PP. The migrating groups are each positioned anti to one another, one group entering whilst the other leaves from the opposite side of the stereocentre. This inverts the configuration at each appropriate centre. Undoubtedly arising from this system is the

234 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition neoclerodane diterpene salvinorin A, the active hallucinogen from Salvia divinorum (Labiatae/ Lamiaceae), a traditional medicine of some Mexican Indians. Salvinorin A is currently attracting a lot of interest, since it is a selective agonist at the kappa-opioid receptor (see page 352). It is the first non-nitrogenous agonist known, and is a valuable lead for the development of other selective ligands that may have therapeutic potential in a range of conditions, including pain, nausea, and depression. SESTERTERPENES (C25 ) Although many examples of this group of natural terpenoids are now known, they are found principally in fungi and marine organisms, and span relatively few structural types. The origins of ophiobolene and ophiobolin A (Figure 5.58) from cyclization of geranylfarnesyl PP

(GFPP) in the plant pathogen Helminthosporium maydis is shown in Figure 5.58, and provides no novel features except for an experimentally demonstrated 1,5-hydride shift. GFPP arises by a continuation of the chain extension process, adding a further IPP unit to GGPP. Ophiobolin A shows a broad spectrum of biological activity against bacteria, fungi, and nematodes. The most common type of marine sesterterpenoid is exemplified by sclarin, and this structure can be envisaged as the result of a concerted cyclization sequence (Figure 5.59) analogous to that seen with GGPP in the diterpenoids, and with squalene in the hopanoids (see below). TRITERPENES (C30 ) Triterpenes are not formed by an extension of the now familiar process of adding IPP to the growing chain. Instead, two molecules of FPP are joined tail-to-tail to yield W–M 1,5-hydride shift generates allylic cation; cyclization onto this cation follows; the cation is eventually quenched by water

concerted cyclizations initiated by allylic cation

H

OPP geranylfarnesyl PP (GFPP)

H

O

H2O H

H

H OHC H O

H

O

O

H

H

O

O H

H

HO

ophiobolene (ophiobolin F)

HO ophiobolin A

Figure 5.58

HO

OPP

O H

AcO H H

H

Figure 5.59

H

H GFPP

sclarin

O

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 235 FPP 1′

E1

PPO

*

2′

3′

1

FPP

* electrophilic addition giving tertiary cation

allylic cation OPP H H * *

loss of proton with formation of cyclopropane ring loss of diphosphate gives primary cation

H * OPP *

*

H

*

H presqualene PP

1,3-alkyl shift generates new cyclopropane ring and more favourable tertiary cation

H W–M 1,3-alkyl shift *

cation quenched by attack of hydride (NADPH) H

*

*

H

* H

H bond cleavage produces alkene and favourable allylic cation E1

NADPH H H * E1: squalene synthase

*

squalene

Figure 5.60 squalene (Figure 5.60); in general, FPP is formed from MVA (see page 192). Squalene is a hydrocarbon originally isolated from the liver oil of shark (Squalus sp.), but was subsequently found in rat liver and yeast, and these systems were used to study its biosynthetic role as a precursor of triterpenes and steroids. Several seed oils are now recognized as quite rich sources of squalene, e.g. Amaranthus cruentus (Amaranthaceae). During the coupling process, which on paper merely requires removal of the two diphosphate groups, a proton from a C-1 position of one molecule of FPP is lost and a proton from NADPH is inserted. Difficulties with formulating a plausible mechanism for this unlikely reaction were resolved when presqualene diphosphate, an intermediate in the process, was isolated from rat liver. Its characterization as a cyclopropane derivative immediately ruled out all the hypotheses current at the time.

The formation of presqualene PP in Figure 5.60, is initiated by attack of the 2,3-double bond of FPP onto the farnesyl cation, which is mechanistically equivalent to normal chain extension using IPP. The resultant tertiary cation is discharged by loss of a proton and formation of a cyclopropane ring, giving presqualene PP. An exactly analogous sequence was used for the origins of irregular monoterpenes (see page 205). Obviously, to then form squalene, C-1s of the two FPP units must eventually be coupled, whilst presqualene PP formation has actually joined C-1 of one molecule to C-2 of the other. To account for the subsequent change in bonding of the two FPP units, a further cyclopropane cationic intermediate is proposed. Loss of diphosphate from presqualene PP would give an unfavourable primary cation, which via Wagner–Meerwein rearrangement can generate a tertiary carbocation and achieve the required C-1–C-1 bond.

236 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

squalene O2, FAD NADPH

E1

sequence of W–M 1,2-hydride and 1,2-methyl shifts H

H

H

cyclizations

3

≡

H

2

HO

O (3S)-2,3-oxidosqualene (squalene oxide)

H

H H HO

H protosteryl cation animals fungi

H protosteryl cation plants

loss of proton gives alkene

loss of proton leads to cyclopropane

H

H H

E1: squalene epoxidase HO

HO

H

H cycloartenol

lanosterol

Figure 5.61 protonation of epoxide allows ring opening to tertiary cation

electrophilic addition gives tertiary cation + 6-membered ring

electrophilic addition gives tertiary cation + 6-membered ring

H HO

O

HO

2,3-oxidosqualene

H

H

H protosteryl cation

Figure 5.62

H

electrophilic addition gives tertiary cation + 5-membered ring electrophilic addition H gives tertiary cation

W–M rearrangement; ring expansion at expense of tertiary → secondary cation

H HO

H

HO

H

H HO

H

H HO

H

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 237 Breaking the original but now redundant C-1–C-2 bond can give an allylic cation, and the generation of squalene is completed by supply of hydride from NADPH. Squalene synthase catalyses all steps in the sequence, the formation of presqualene PP from FPP, its subsequent rearrangement, and NADPH-dependent reduction. Cyclization of squalene is via the intermediate 2,3-oxidosqualene (squalene-2,3-oxide; Figure 5.61), produced in a reaction catalysed by squalene epoxidase, a flavoprotein requiring O2 and NADPH cofactors. If oxidosqualene is suitably positioned and folded on the enzyme surface, then the polycyclic triterpene structures formed can be rationalized in terms of a series of cyclizations, followed by a sequence of concerted Wagner–Meerwein migrations of hydride and methyl groups (Figure 5.61). The cyclizations are carbocation-mediated and proceed in a stepwise sequence (Figure 5.62). Thus, protonation of the epoxide group will allow opening of this ring and generation of the preferred tertiary carbocation, suitably placed to allow electrophilic addition to a double bond, formation of a six-membered ring, and production of a new tertiary carbocation. This process continues twice more, generating the preferred tertiary carbocation (Markovnikov addition) after each ring formation, though the third ring formed is consequently a five-membered one. This is expanded to a six-membered ring via a Wagner–Meerwein 1,2-alkyl shift, resulting in some relief of ring strain, though sacrificing a tertiary carbocation for a secondary one. A further electrophilic addition generates the tertiary protosteryl cation (Figure 5.62). The stereochemistries in this cation are controlled by the type of folding achieved on the enzyme surface, and this probably also limits the extent of the cyclization process. Thus, if the folded oxidosqualene approximates to a chair–boat–chair–boat conformation (Figure 5.63), the transient protosteryl cation will be produced with these conformational characteristics. This cation then undergoes a series of Wagner–Meerwein 1,2-shifts, first migrating a hydride and generating a new cation, migrating the next hydride, then a methyl and so on until a proton is lost forming a double bond and thus creating lanosterol (Figure 5.63). The stereochemistry of the protosteryl cation in Figure 5.63 shows how favourable this sequence will be, and emphasizes that, in the ring system, the migrating groups are positioned anti to each other, one group entering whilst the other leaves from the opposite side of the stereocentre (compare diterpenes, page 233). This, of course, inverts configurations at each appropriate centre. No anti group is available to migrate to C-9 (steroid numbering), and the reaction terminates by loss of the proton H-9. Lanosterol is

a typical animal triterpenoid, and the precursor for cholesterol and other sterols in animals (see page 248) and fungi (see page 254). In plants, its intermediate role is taken by cycloartenol (Figure 5.63), which contains a cyclopropane ring, generated by inclusion of carbon from the methyl at C-10. For cycloartenol, H-9 is not lost, but migrates to C-8, and the carbocation so formed is quenched by cyclopropane formation and loss of one of the methyl protons. For many plant steroids, this cyclopropane ring has then to be reopened (see page 251). Most natural triterpenoids and steroids contain a 3-hydroxyl group, the original epoxide oxygen from oxidosqualene. An additional feature of the protosteryl cation is that the C-10 methyl and H-5 also share an anti-axial relationship, and are also susceptible to Wagner–Meerwein rearrangements, so that the C-9 cation formed in the cycloartenol sequence may then initiate further migrations. This can be terminated by formation of a 5,6-double bond (Figure 5.64), as in the pathway to the cucurbitacins, a group of highly oxygenated triterpenes encountered in the Cucurbitaceae, the cucumber/melon/marrow family. These compounds are characteristically bitter tasting, purgative, and extremely cytotoxic. Should oxidosqualene be folded in a roughly chair–chair–chair–boat conformation by binding to another type of cyclase enzyme (Figure 5.65), then an identical carbocation mechanism ensues. However, the transient dammarenyl cation formed has different stereochemical features to the protosteryl cation. Whilst a series of Wagner–Meerwein migrations can occur, there is relatively little to be gained on purely chemical grounds, since these would invert stereochemistry and destroy what is already a very favourable conformation. Instead, the dammarenyl cation typically undergoes further carbocation-promoted cyclizations, without any major changes to the ring system already formed. Occasionally, the migrations do occur: euphol (Figure 5.65) from Euphorbia species (Euphorbiaceae) is a stereoisomer of lanosterol. If the Wagner–Meerwein rearrangements do not take place, then the dammarenyl cation could be quenched with water (Figure 5.66), giving the epimeric dammarenediols, as found in Dammar resin from Balanocarpus heimii (Dipterocarpaceae) and ginseng (Panax ginseng; Araliaceae) [Box 5.11]. Alternatively, the alkyl migration shown gives the baccharenyl cation, relieving some ring strain by creating a six-membered ring, despite sacrificing a tertiary carbocation for a secondary one. A pentacyclic ring system can now be formed by cyclization onto the double bond, giving a new five-membered ring and the tertiary lupenyl cation. Although this appears to

238 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition H

H O HO

H

E1 E2

chair – boat – chair – boat

H

H

2,3-oxidosqualene H 2

HO

3

4

6

5

H

9

10

1

7

H

8

H protosteryl cation

H

HO

H H

sequence of W–M 1,2-hydride and 1,2-methyl shifts

H sequence of W–M 1,2-hydride and 1,2-methyl shifts

E1 H

H

H

H

H

H

2

HO

H

H cyclopropane ring formation and loss of proton from C-10 methyl

HO

3

1 4

9

10 6

5

7

H

8

H protosteryl cation

E1

loss of H-9 creates double bond

H

H

E2

H

H HO

H H

HO

H H

≡

≡ H

H H

HO

HO

H cycloartenol

H lanosterol

E1: oxidosqualene:cycloartenol cyclase (cycloartenol synthase) E2: oxidosqualene:lanosterol cyclase (lanosterol synthase)

Figure 5.63 contradict the reasoning used above for the dammarenyl → baccharenyl transformation, the contribution of the enzyme involved must also be considered in each case. A five-membered ring is not highly strained, as evidenced by all the natural examples encountered. Loss of a proton from the lupanyl cation gives lupeol, found in lupin (Lupinus luteus; Leguminosae/Fabaceae). Ring expansion in the lupanyl cation by bond migration gives the oleanyl system, and labelling studies have demonstrated this ion is discharged by hydride migrations and loss

of a proton, giving the widely distributed β-amyrin. Formation of the isomeric α-amyrin involves first the migration of a methyl in the oleanyl cation, then discharge of the new taraxasteryl cation by three hydride migrations and loss of a proton. Loss of a proton from the non-migrated methyl in the taraxasteryl cation is an alternative way of achieving a neutral molecule, and yields taraxasterol found in dandelion (Taraxacum officinale; Compositae/Asteraceae). Comparison with α-amyrin shows the subtly different stereochemistry

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 239 first sequence of W–M 1,2-hydride and 1,2-methyl shifts

H

H

H H

10 5

HO

further sequence of W–M 1,2-methyl and 1,2-hydride shifts, terminated by loss of proton

H E1

HO

H

H

protosteryl cation

H E1

OH H

O H

HO

H

OAc

various oxidative modifications

5

HO

O

H

H

O

OH

H

cucurbitacin E

6

cucurbitadienol

E1: cucurbitadienol synthase (2,3-oxidosqualene is substrate)

Figure 5.64

O chair – chair – chair – boat 2,3-oxidosqualene

H

H

H

≡

H

HO H

H

H HO

sequence of W–M 1,2-hydride and 1,2-methyl shifts terminated by loss of proton

H dammarenyl cation

H H

H

≡

HO H

H

HO

H euphol

Figure 5.65

lanosterol

240 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition ring expansion at expense of tertiary → secondary cation

OH cation quenched by H2O

H H

HO

H

E1 HO

H

H dammarenediol

dammarenyl cation W–M 1,2-alkyl shift

E2 E3

ring expansion at expense of tertiary → secondary cation

5-membered ring formation gives tertiary cation

H

H

H H

E2 E3

H HO

H

H HO

baccharenyl cation

H

≡

lupeol

W–M 1,2-methyl shift

three 1,2-hydride shifts, then loss of proton; note inversions of stereochemistry at C-18/19 H H H H H H

HO

H oleanyl cation – H+

– H+ E3 H

18

H taraxasteryl cation W–M 1,2-hydride shifts

– H+

19 18

19 18

H

H

H

H

H HO

H

H

H

oleanyl cation

HO

H

1,2-methyl shift allows secondary → tertiary cation

E3

H

HO

W–M 1,2-hydride shifts

E2

H

W–M 1,2alkyl shift

H

H

HO

H HO

– H+

lupanyl cation

two 1,2-hydride shifts then loss of proton; note inversion of H stereochemistry H H at C-18 H

both 20S and 20R dammarendiols are known, so attack may occur from either side of cation

H

H2O

H

20S

H

H

β-amyrin E1: dammarenediol-II synthase E2: oxidosqualene:lupeol cyclase (lupeol synthase)

H HO

taraxasterol

H α-amyrin

E3: oxidosqualene:β-amyrin cyclase (β-amyrin synthase) (2,3-oxidosqualene is substrate for enzymes)

Figure 5.66 present, because the inversions of configuration caused by hydride migrations have not occurred. Where evidence is available, these extensive series of cyclizations and Wagner–Meerwein rearrangements appear to be catalysed by a single enzyme which converts oxidosqualene into the final product, e.g. lanosterol, cycloartenol, lupeol, or β-amyrin. Enzymes can be monofunctional, synthesizing

a single product, or in some cases the triterpenoid cyclase is multifunctional, catalysing formation of a range of structurally related products, e.g. lupeol, α-amyrin, taraxasterol, and others. Remarkably, changing a single amino acid residue in lupeol synthase from olive (Olea europaea; Oleaceae) was sufficient to alter the protein’s function; it became instead a producer of β-amyrin.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 241

H

17

17

H

H

H

H HO

17

H

H

HO lupeol

H

O

H HO

H

α-amyrin

β-amyrin

O

O

H H

H

R H

H HO

H

CO2H

HO

H

H HO

H

H

ursolic acid

R = CH2OH, betulin R = CO2H, betulinic acid

CO2H

oleanolic acid CO2H

H

O

H O HO2C

H

CO2H H

H O

HO

H bevirimat

H CHO quillaic acid

CO2H

H

OH

H HO

H glycyrrhetic acid

Figure 5.67 Further modification of these hydrocarbons to triterpene acids is frequently encountered (Figure 5.67). Olives (Olea europaea; Oleaceae) contain large quantities of oleanolic acid, in which the C-17 methyl of β-amyrin has been oxidized to a carboxylic acid. The leaves of olive contain a mixture of oleanolic acid and betulinic acid, the latter an oxidized version of lupeol. The corresponding derivative of α-amyrin is ursolic acid, found in bearberry (Arctostaphylos uva-ursi; Ericaceae). These are presumably produced by cytochrome P-450-dependent oxidations, but details are not known. Oxidative transformations at other methyl groups and/or ring carbon atoms are required to produce triterpenoids such as glycyrrhetic acid and quillaic acid (see triterpenoid saponins below). The alcohol betulin is a major component in the bark of white birch (Betula alba; Betulaceae), where it comprises up to 24% of the outer bark layer; large amounts of the rarer betulinic acid may be obtained by selective chemical oxidation of betulin. Derivatives of betulinic

acid and betulin are currently attracting considerable interest in HIV therapeutics by inhibiting viral growth in a manner different from that of current HIV drugs. The most promising compound, currently in clinical trials, is the semi-synthetic 3 ,3 -dimethylsuccinyl ester bevirimat (Figure 5.67). Bacterial membranes frequently contain hopanoids (Figure 5.68), triterpenoid compounds that appear to take the place of the sterols that are typically found in the membranes of higher organisms, helping to maintain structural integrity and to control permeability. Hopanoids are also the characteristic triterpenes in ferns. Hopanoids arise from squalene by a similar carbocation cyclization mechanism, but do not involve the initial epoxidation to oxidosqualene. Instead, the carbocation is produced by protonation (compare the cyclization of GGPP to copalyl PP, page 228), and the resultant compounds tend to lack the characteristic 3-hydroxyl group, e.g. hopene from

242 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

cyclization is initiated by protonation of double bond to give tertiary cation squalene

H E1

E2

hopanyl cation E2 E1

E1 OH

H

H

H H

OH

H

H hopan-22-ol (diplopterol)

H

H

H

H

H H

H

H

H HO hopene

H

≡

H H

tetrahymanol

E1: squalene:hopene cyclase E2: squalene:tetrahymanol cyclase

Figure 5.68 Alicyclobacillus acidocaldarius (Figure 5.68). On the other hand, tetrahymanol from the protozoan Tetrahymena pyriformis, because of its symmetry, might appear to have a 3-hydroxyl group, but this is derived from water and not molecular oxygen, as would be the case if oxidosqualene were involved. As in formation of the protosteryl cation (page 237), Markovnikov additions followed by Wagner–Meerwein ring expansions (rather than anti-Markovnikov additions) may occur during the cyclization mechanisms shown in Figure 5.68. Triterpenoid Saponins The pentacyclic triterpenoid skeletons exemplified by lupeol, α-amyrin, and β-amyrin (Figure 5.67) are frequently encountered in the form of triterpenoid saponin structures. Saponins are glycosides

which, even at low concentrations, produce a frothing in aqueous solution, because they have surfactant and soap-like properties. The name comes from the Latin sapo, meaning soap, and plant materials containing saponins were originally used for cleansing clothes, e.g. soapwort (Saponaria officinalis; Caryophyllaceae) and quillaia or soapbark (Quillaja saponaria; Rosaceae). These materials also cause haemolysis, lysing red blood cells by increasing the permeability of the plasma membrane, and thus they are highly toxic when injected into the bloodstream. Some saponin-containing plant extracts have been used as arrow poisons. However, saponins are relatively harmless when taken orally, and some of our valuable food materials, e.g. beans, lentils, soybeans, spinach, and oats, contain significant amounts. Sarsaparilla (see page 264) is rich in steroidal saponins, but is widely used in the manufacture of non-alcoholic drinks. Toxicity is

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 243 29

30

O

20 19 18 13

12 1 2

A

25

9 10 5

3

B

26

C

14

8 7

4 24

11

E 17

D

21

O

22

H

28

O

16 15

H

27

6 23

pentacyclic triterpenoid skeleton

HO

O

H O

potential sites for oxidation (β-amyrin type)

Figure 5.69 minimized during ingestion by low absorption, and by hydrolysis. Acid-catalysed hydrolysis of saponins liberates sugar(s) and an aglycone (sapogenin) which can be either triterpenoid or steroidal (see page 259) in nature. Some plants may contain exceptionally high amounts of saponins, e.g. about 10% in quillaia bark. Triterpenoid saponins are rare in monocotyledons, but abundant in many dicotyledonous families. Several medicinally useful examples are based on the β-amyrin subgroup (Figure 5.69), and many of these possess carboxylic acid groups derived by oxidation of methyl groups, those at positions 4 (C-23), 17 (C-28) and 20 (C-30) on the aglycone ring system being subject to such oxidation. Less oxidized formyl (–CHO) or hydroxymethyl (–CH2 OH) groups may be encountered, and positions 11 and 16 may also be oxygenated. Sugar

residues are usually attached to the 3-hydroxyl, with one to six monosaccharide units, the most common being glucose, galactose, rhamnose, and arabinose, with uronic acid units (glucuronic acid and galacturonic acid) also featuring (see page 488). Thus, quillaia bark contains a saponin mixture with quillaic acid (Figure 5.67) as the principal aglycone [Box 5.11]. The medicinally valuable root of liquorice (Glycyrrhiza glabra; Leguminosae/Fabaceae) contains glycyrrhizin, a mixture of potassium and calcium salts of glycyrrhizic acid (Figure 5.70), which is composed of the aglycone glycyrrhetic acid and two glucuronic acid units [Box 5.11]. Ginseng is a herbal drug derived from the roots of Panax ginseng (Araliaceae) that is widely held to counter stress and improve general well-being. A group of saponins based on the dammarane skeleton and termed ginsenosides (see Figure 5.72) are most likely the biologically active components [Box 5.11].

Box 5.11 Liquorice Liquorice (licorice; glycyrrhiza) is the dried unpeeled rhizome and root of the perennial herb Glycyrrhiza glabra (Leguminosae/Fabaceae). A number of different varieties are cultivated commercially, including G. glabra var. typica (Spanish liquorice) in Spain, Italy, and France, and G. glabra var. glandulifera (Russian liquorice) in Russia. Russian liquorice is usually peeled before drying. G. uralensis (Manchurian liquorice) from China is also commercially important. Much of the liquorice is imported in the form of an extract, prepared by extraction with water, then evaporation to give a dark black solid. Most of the liquorice produced is used in confectionery and for flavouring, including tobacco, beers, and stouts. Its pleasant sweet taste and foaming properties are due to saponins. Liquorice root contains about 20% of water-soluble extractives, and much of this (typically 3–5% of the root, but up to 12% in some varieties) is comprised of glycyrrhizin, a mixture of the potassium and calcium salts of glycyrrhizic (=glycyrrhizinic) acid (Figure 5.70). Glycyrrhizic acid is a diglucuronide of the aglycone glycyrrhetic (=glycyrrhetinic) acid. The bright yellow colour of liquorice root is provided by flavonoids (1–1.5%), including liquiritigenin and isoliquiritigenin and their corresponding glucosides (see page 169). Considerable amounts (5–15%) of sugars (glucose and sucrose) are also present. Glycyrrhizin is reported to be 50–150 times as sweet as sucrose, and liquorice has thus long been used in pharmacy to mask the taste of bitter drugs. Its surfactant properties have also been exploited in various formulations, as have its demulcent

244 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.11 (continued) CO2H CO2

O D-glucuronic acid CO2H HO HO CO2H

HO HO

H O

H

O O

H O

H

O

O2C

O OH β1→2 D-glucuronic acid

H 2Na

O

glycyrrhetic acid

H carbenoxolone sodium

glycyrrhizic acid

Figure 5.70

O D-fucose O D-glucuronic acid CO2H D-xylose O O HO HO O HO OH HO OH

H H O

O O

HO OH D-galactose

H CHO quillaic acid

CO2

OH

O

O

O

O

OH

O OH L-rhamnose

OH HO O

OH

O

OR HO OH

O

OH L-arabinose

D-xylose HO

OH

O

QS-21api, R = D-apiose D-apiose QS-21xyl, R = D-xylose

O

HO

HO

OH OH

O

OH

D-xylose

Figure 5.71 and mild expectorant properties. More recently, some corticosteroid-like activity has been recognized, with liquorice extracts displaying mild anti-inflammatory and mineralocorticoid activities. These have been exploited in the treatment of rheumatoid arthritis, Addison’s disease (chronic adrenocortical insufficiency), and various inflammatory conditions. Glycyrrhetic acid has been implicated in these activities, and has been found to inhibit enzymes that catalyse the conversion of prostaglandins and glucocorticoids into inactive metabolites. This results in increased levels of prostaglandins, e.g. PGE2 and PGF2α (see page 60), and of hydrocortisone (see page 278). A semi-synthetic derivative of glycyrrhetic acid, the hemisuccinate carbenoxolone sodium (Figure 5.70), has been widely prescribed for the treatment of gastric ulcers, and also duodenal ulcers. Because of side-effects, typically loss of potassium and increase in sodium levels, it has been superseded by newer drugs.

Quillaia Quillaia bark or soapbark is derived from the tree Quillaja saponaria (Rosaceae) and other Quillaja species found in Chile, Peru, and Bolivia. The bark contains up to 10% saponins, a mixture known as ‘commercial saponin’ which is used as a foaming agent

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 245

Box 5.11 (continued) in beverages and emulsifier in foods. Quillaia’s surfactant properties are occasionally exploited in pharmaceutical preparations, where in the form of quillaia tincture it is used as an emulsifying agent, particularly for fats, tars, and volatile oils. The bark contains a mixture of saponins which on hydrolysis liberates quillaic acid (Figure 5.67) as the aglycone, together with sugars, uronic acids, and acids from ester functions. Saponins from quillaia are also showing great promise as immunoadjuvants, substances added to vaccines and other immunotherapies designed to enhance the body’s immune response to the antigen. One such agent, QS-21A (Figure 5.71), is a mixture of the two saponins QS-21api and QS-21xyl , each incorporating a quillaic acid triterpenoid core, flanked on either side by complex oligosaccharides, one of which includes a chain comprised of two fatty acyl components. These compounds were isolated from the 21st chromatographic fraction of the Q. saponaria extract.

Ginseng The roots of the herbaceous plants Panax ginseng (Araliaceae) from China, Korea and Russia, and related Panax species, e.g. P. quinquefolium (American ginseng) from the USA and Canada and P. notoginseng (Sanchi-ginseng) from China, have been widely used in China and Russia for the treatment of a number of diseases, including anaemia, diabetes, gastritis, insomnia, sexual impotence, and as a general restorative, promoting health and longevity. Interest in the drug has increased considerably in recent years, and ginseng is widely available as a health food in the form of powders, extracts, and teas. The dried and usually peeled root provides white ginseng, whereas red ginseng is obtained by steaming the root, this process generating a reddish-brown caramel-like colour, and reputedly enhancing biological activity. Ginseng is classified as an ‘adaptogen’, an agent that helps the body to adapt to stress, improving stamina and concentration, and providing a normalizing and restorative effect. It is also widely promoted as an aphrodisiac. The Korean root is highly prized and the most expensive. Long-term use of ginseng can lead to symptoms similar to those of corticosteroid poisoning, including hypertension, nervousness, and sleeplessness in some subjects, yet hypotension and tranquillizing effects in others. The benefits of ginseng treatment are by no means confirmed at the pharmacological level, though ginseng does possess antioxidant activity, can affect both central nervous system and neuroendocrine functions, can alter carbohydrate and lipid metabolism, and modulates immune function. Many of the secondary metabolites present in the root have now been identified. It contains a large number of triterpenoid saponins based on the dammarane subgroup, saponins that have been termed ginsenosides by Japanese investigators, or panaxosides by Russian researchers. These are derivatives of two main aglycones, protopanaxadiol and protopanaxatriol (Figure 5.72), though the aglycones liberated on acid hydrolysis are panaxadiol and panaxatriol respectively. Acid-catalysed cyclization in the side-chain produces an ether ring (Figure 5.72). Sugars are present in the saponins on the 3and 20-hydroxyl groups in the diol series, and the 6- and 20-hydroxyl groups in the triol series. Over 30 ginsenosides have been characterized from the different varieties of ginseng, with ginsenoside Rb1 (Figure 5.72) of the diol series typically being the most abundant constituent. Ginsenoside Rg1 (Figure 5.72) is usually the major component representative of the triol series. Some other variants are shown in Figure 5.72. Particularly in white ginseng, many of the ginsenosides are also present as esters with malonic acid. Steaming to prepare red ginseng causes partial hydrolysis of esters and glycosides; there is undoubtedly greater antioxidant activity in the steamed product, though this is derived mainly from various phenolic constituents. Ginsenosides Rb1 and Rg1 appear to be the main representatives in P . ginseng, ginsenosides Rb1 , Rg1 , and Rd in P. notoginseng, and ginsenosides Rb1 , Re, and malonylated Rb1 in P. quinquefolium. The pentacyclic triterpenoid sapogenin oleanolic acid (Figure 5.67) is also produced by hydrolysis of the total saponins of P. ginseng, and is present in some saponin structures (chikusetsusaponins). The saponin contents of P . notoginseng (about 12%) and P. quinquefolium (about 6%) are generally higher than that of P. ginseng (1.5–2%). Pharmacological studies on individual ginsenosides are being facilitated by development of enzymic procedures for interconverting the structures, e.g. by selective hydrolysis of sugar groups, or by selective glycosylations. The aglycone mixture of protopanaxadiol and protopanaxatriol (pandimex®) is being tested clinically as an anticancer agent. The root of Eleutherococcus senticosus (Acanthopanax senticosus) (Araliaceae) is used as an inexpensive substitute for ginseng, and is known as Russian or Siberian ginseng. This material is held to have adaptogenic properties similar to P. ginseng, and a number of eleutherosides have been isolated. However, the term eleutheroside has been applied to compounds of different chemical classes, and the main active anti-stress constituents appear to be lignan glycosides, e.g. eleutheroside E (≡syringaresinol diglucoside; Figure 5.72) (compare page 152) and phenylpropane glycosides, e.g. eleutheroside B (≡syringin). The leaves of Russian ginseng contain a number of saponins based on oleanolic acid (Figure 5.67), but these are quite different to the ginsenosides/panaxosides found in Panax. Whilst there is sufficient evidence to support the beneficial adaptogen properties for E. senticosus, detailed pharmacological confirmation is not available.

246 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.11 (continued)

OGly2 HO

20

H

protonation of double bond gives tertiary cation and allows formation of ether by attack of hydroxyl

OH HO

H

H

20

HO H

H+ Gly1O

H HO

H ginsenoside (panaxoside) Gly1

Gly2

ginsenoside Rb1

β1→2 Glc–Glc–

β1→6 –Glc–Glc

ginsenoside Rb2

β1→2 Glc–Glc–

β1→6 –Ara–Glc

ginsenoside Rc

β1→2 Glc–Glc–

H (20S)-protopanaxadiol

H

O

β1→6 –Ara–Glc

HO

H

H

20

H

H

20

O HO H

H+ H

panaxadiol

OH HO

H HO

6

OGly1 ginsenoside (panaxoside)

H

20R

H

H+

H HO

H

Glc

OGly2 HO

20R

H

H

(furanose)

ginsenoside Rd

H

H HO

both 20R- and 20S-panaxadiol are formed, with the 20R-epimer predominating; since both isomers are produced, this implies an equilibration, probably through loss of H2O and generation of a carbocation at C-20 in protopanaxadiol HO

(pyranose)

β1→2 Glc–Glc–

H

H+

H

3

OH

H HO

H

H

OH (20S)-protopanaxatriol

OH panaxatriol

as mixture of 20R and 20S epimers; see notes on panaxadiol above

Gly1

Gly2

ginsenoside Re

β1→2 Rha–Glc–

Glc

ginsenoside Rf

β1→2 Glc–Glc–

H

ginsenoside Rg1

Glc

Glc

GlcO

ginsenoside Rg2

β1→2 Rha–Glc–

H

MeO

eleutherosides in Eleutherococcus senticosus OMe OMe

GlcO O MeO H

H OMe O

OH syringin (eleutheroside B)

Figure 5.72

OGlc syringaresinol diglucoside (eleutheroside E)

OMe

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 247 21 18 12

H 2

H

H

19 11 9

1

3

HO

A

10

4

cholesterol

A

25

D

26

H

27

16

8

A

7

5

B

all-trans

H C D A

H

H

H C D H

B

A

C

D

B

H H A ring aromatic

H

A/B cis

D

H

H

H

6

C

B

steroid numbering

H

A/B cis, C/D cis H

H C

D

B

A

24

15

H H

23

17 13 14

C

B

22

20

A

H

H

C

B

A

H

H Δ4-unsaturation

Δ5-unsaturation H

H H

H trans-decalin

D

ring flip in both rings

H

H

C

B

D

H H Δ5,7-unsaturation

H

≡

H

rotate 60˚ cis-decalin

Figure 5.73 STEROIDS

Stereochemistry and Nomenclature

The steroids are modified triterpenoids containing the tetracyclic ring system of lanosterol (Figure 5.61), but lacking the three methyl groups at C-4 and C-14. Cholesterol (Figure 5.73) typifies the fundamental structure, but further modifications, especially to the side-chain, help to create a wide range of biologically important natural products, e.g. sterols, steroidal saponins, cardioactive glycosides, bile acids, corticosteroids, and mammalian sex hormones. Because of the profound biological activities encountered, many natural steroids and a considerable number of synthetic and semi-synthetic steroidal compounds are routinely employed in medicine. The markedly different biological activities observed emanating from compounds containing a common structural skeleton are in part ascribed to the functional groups attached to the steroid nucleus, and in part to the overall shape conferred on this nucleus by the stereochemistry of ring fusions.

Ring systems containing six-membered or five-membered rings can be trans-fused as exemplified by trans-decalin or cis-fused as in cis-decalin (Figure 5.73). The trans-fusion produces a flattish molecule when two chair conformations are present. The only conformational mobility allowed is to less favourable boat forms. Bridgehead hydrogen atoms (or other substituents) are axial to both of the rings. In contrast, the cis-fused decalin is basically a bent molecule; it is found to be flexible, in that alternative conformers are possible, with both rings still being in chair form. Bridgehead substituents are axial to one ring, whilst being equatorial to the other, in each conformer. However, this conformational flexibility will be lost if either ring is then fused to a third ring, so is not encountered in steroid structures In natural steroids, there are examples of the A/B ring fusion being trans or cis, or having unsaturation, either

248 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition other groups are added as prefixes. Stereochemistry of substituents is represented by α (on the lower face of the molecule when it is drawn according to customary conventions as in Figure 5.74), or β (on the upper face). Ring fusions may be designated by using α or β for the appropriate bridgehead hydrogen, particularly those at positions 5 and 14, which will define the A/B and C/D fusions respectively, e.g. 5β-cholestane has the A/B rings cis-fused. Since the parent hydrocarbon assumes that ring fusions are trans, the stereochemistry for ring fusions is usually only specified where it is cis. Cholesterol is thus cholest-5-en-3β-ol. The term nor is affixed to indicate loss of a carbon atom, e.g. 19-norsteroids (see page 288) lack C-19 (the methyl at C-10).

4 or 5 . In some compounds, notably the oestrogens, ring A can even be aromatic; clearly, there can then be no bridgehead substituent at C-10 and, therefore, the normal C-10 methyl (C-19) must be lost. All natural steroids have a trans B/C fusion, though cis forms can be made synthetically. The C/D fusion is also usually trans, though there are notable exceptions, such as the cardioactive glycosides. Of course, such comments apply equally to some of the triterpenoid structures already considered. However, it is in the steroid field where the relationship between stereochemistry and biological activity is most marked. The overall shapes of some typical steroid skeletons are shown in Figure 5.73. Systematic steroid nomenclature is based on a series of parent hydrocarbons, including gonane, estrane, androstane, pregnane, cholane, cholestane, ergostane, campestane, stigmastane, and poriferastane (Figure 5.74). The triterpenoid hydrocarbons lanostane and cycloartane are similarly used in systematic nomenclature and are also included in Figure 5.74. It is usual to add only unsaturation (ene/yne) and the highest priority functional group as suffixes to the root name;

Cholesterol In animals, the triterpenoid alcohol lanosterol (C30 ) is converted into cholesterol (C27 ) (Figure 5.75), a process that, as well as the loss of three methyl groups, requires reduction of the side-chain double bond, and generation of a 5,6 double bond in place of the 8,9 double bond. The sequence of these steps is, to some extent, variable and

H H

H H

H H

H H

H

H H

H

H gonane (C17)

H H

H

H

H androstane (C19)

estrane (C18)

H

pregnane (C21)

241

24

H H

H

H

H

H

H

cholestane (C27)

H campestane (C28)

24

H

H H

H

H

H H

H

H

Figure 5.74

H

242

24

stigmastane (C29)

H

241

24

H

H

ergostane (C28)

242 1

H

H

H

H cholane (C24)

24

H

H

30

30

H

H poriferastane (C29)

29

28

lanostane (C30)

241

29

28

cycloartane (C30)

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 249

H H H HO

5

9

H H

14

8

H 4

HO

6

H

cholesterol

HO

H

lanosterol

cycloartenol

Figure 5.75

sequential oxidation of 14-methyl group to aldehyde R 14

R

O2 NADPH

peroxy adduct formation via nucleophilic attack of peroxy-enzyme onto carbonyl R

O2 NADPH

D E1

Enz–Fe–OOH E1

E1

15

O

OH

R

14α-formyl derivative

homolytic cleavage of peroxy bond

O

O H

O Fe–Enz

reduction of double bond R

R

NADPH H2O E2

H

H

E1

H E1: sterol 14-demethylase E2: sterol Δ14-reductase

elimination, formate as leaving group

R

Δ8,14-diene

R

H H O Fe–Enz HCO2H methyl group lost as formic acid

O

O H O Fe–Enz

Figure 5.76 dependent on the organism involved. Accordingly, these individual transformations are considered rather than the overall pathway. The methyl at C-14 is usually the one lost first, and this is removed as formic acid. The reaction is catalysed by a cytochrome P-450 monooxygenase which achieves two oxidation reactions to give the 14α-formyl derivative (Figure 5.76), and loss of this formyl group giving the 8,14 diene, most probably via homolytic cleavage of the peroxy adduct as indicated (compare similar peroxy adducts and mechanisms involved in side-chain cleavage from ring D, page 292, and in A ring aromatization, page 292). The 14-demethyl sterol is then obtained by an NADPH-dependent reduction step, the 15-proton being derived from water. Loss of the C-4 methyl groups occurs sequentially, usually after removal of the 14α-methyl. Both carbon atoms are oxidized to carboxyl groups, then cleaved off

via a decarboxylation mechanism (Figure 5.77). This is facilitated by oxidizing the 3-hydroxyl to a ketone, thus generating intermediate β-keto acids. In this sequence, the enolate is restored to a ketone in which the remaining C-4 methyl takes up the more favourable equatorial (4α) orientation. In animals and yeasts, the two methyl groups are removed by the same enzyme systems; plants are found to employ a different set of enzymes for the removal of the α- and β-methyl groups. The side-chain 24 double bond is reduced by an NADPH-dependent reductase, hydride from the coenzyme being added at C-25, with H-24 being derived from water (Figure 5.78). The 8 double bond is effectively migrated to 5 via 7 and the 5,7 diene (Figure 5.79). This sequence involves an allylic isomerization, a dehydrogenation, and a reduction. Newly introduced protons at C-9 and C-8 originate from water, and that at C-7 from NADPH.

250 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition sequential oxidation of 4a-methyl group to carboxyl O2 NADPH

oxidation of 3b-alcohol to ketone NAD+

A HO

4

HO HO2C

E1

H

decarboxylation of b-keto acid – CO2

H

E2

H

O O

HO

H

H enol–keto tautomerism; methyl takes up more favoured equatorial configuration

O H repeat of whole process HO

E1, E2, E3

H

NADPH

H

E3

HO

O

H

H H

reduction of ketone back to 3b-alcohol E1+E2+E3: sterol 4α-methyl oxidase (sterol C-4 demethylase) E1: methylsterol monooxygenase

O

E2: sterol-4α-carboxylate 3-dehydrogenase (decarboxylating) E3: 3-ketosteroid reductase

Figure 5.77

H

modifications outlined above, a further mechanism to reopen the cyclopropane ring is necessary. This is shown in Figure 5.80. Acid-catalysed ring opening is the most likely mechanism, and this can be achieved non-enzymically, but under severe conditions. It is suggested, therefore, that a nucleophilic group from the enzyme attacks C-9, opening the cyclopropane ring and incorporating a proton from water. A trans elimination then generates the 8 double bond. The stereochemistry at C-8 (Hβ) is unfavourable for a concerted mechanism involving loss of H-8 with cyclopropane ring opening. The cyclopropane ring-opening process seems to occur only with 4α-monomethyl sterols. In plants, removal of the first 4-methyl group (4α; note the remaining 4β-methyl group then takes up the α-orientation as in Figure 5.77) is also known to precede loss of the 14α-methyl. Accordingly, the general substrate shown in Figure 5.80 has both 4α- and 14α-methyl groups;

NADPH H2O

25 24

H

H

E1

E1: sterol Δ24-reductase

Figure 5.78

The role of lanosterol in non-photosynthetic organisms (animals, fungi) is taken in photosynthetic organisms (plants, algae) by the cyclopropane triterpenoid cycloartenol (Figure 5.75). This cyclopropane feature is found in a number of plant sterols, though the majority of plant steroids contain the typical C-10 methyl group. This means that, in addition to the lanosterol → cholesterol

9 5

H

B 6

dehydrogenation gives conjugated diene O2 NADPH

allylic isomerization

H 8

7

H H

H

H

Δ7-ene E1: sterol Δ8-Δ7-isomerase (cholestenol Δ-isomerase) E2: Δ7-sterol Δ5-dehydrogenase (lathosterol oxidase)

Figure 5.79

H

NADPH H

H

H

E1

reduction of double bond

E2

H Δ5,7-diene

E3

H

H

E3: sterol Δ7-reductase (7-dehydrocholesterol reductase)

H H

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 251 cyclopropane ring opening initiated by attack of nucleophilic group on enzyme

HO

R

H 9

4

H

Enz

R H

14

8

Enz

E1 HO

R trans elimination E1 HO

4α-monomethyl cycloartanol derivative

4α-monomethyl lanosterol derivative

E1: cycloeucalenol cycloisomerase (cycloeucalenol-obtusifoliol isomerase)

Figure 5.80 side-chain alkylation (see page 252) also commonly precedes these other modifications. The specificity of the cyclopropane ring-opening enzyme means cycloartenol is not converted into lanosterol, and lanosterol is thus absent from virtually all plant tissues. Cholesterol [Box 5.12] is almost always present in plants, though in only trace amounts, and is formed via cycloartenol.

Phytosterols The major sterol found in mammals is the C27 compound cholesterol, which acts as a precursor for other steroid structures such as sex hormones and corticosteroids. The main sterols in plants, fungi, and algae are characterized by extra one-carbon or two-carbon substituents on

Box 5.12 Cholesterol Cholesterol (Figure 5.75) is the principal animal sterol, and since it is a constituent of cell membranes has been found in all animal tissues. It maintains membrane fluidity, microdomain structure, and permeability. It functions as a precursor for steroid hormones (pages 279, 291), bile acids (page 276), vitamin D (page 257), and lipoproteins, but is also correlated with cardiovascular disease, atherosclerosis, hypercholesterolaemia, and gallstone disease. Human gallstones are almost entirely composed of cholesterol precipitated from the bile. Although the processes involved are quite complex, there appears to be a clear correlation between human blood cholesterol levels and heart disease. Atherosclerosis is a hardening of the arteries caused by deposition of cholesterol, cholesterol esters, and other lipids in the artery wall, causing a narrowing of the artery and, thus, an increased risk of forming blood clots (thrombosis). Normally, most of the cholesterol serves a structural element in cell walls, whilst the remainder is transported via the blood and is used for synthesis of steroid hormones, vitamin D, or bile acids. Transport of cholesterol is facilitated by formation of lipoprotein carriers, comprising protein and phospholipid shells surrounding a core of cholesterol, in both free and esterified forms. Low density lipoproteins (LDLs) carry larger amounts of cholesterol and deposit it around the body. High-density lipoproteins (HDLs) pick up free cholesterol and return it to the liver to be degraded. Risk of atherosclerosis increases with increasing levels of LDL cholesterol, and is reduced with increasing levels of HDL cholesterol. Blood LDL cholesterol levels are thus a good statistical indicator of the potential risk of a heart attack. Current recommendations are that total cholesterol levels in blood should be lower than 4 mmol/l, of which LDL cholesterol concentration should be less than 2 mmol/l. The risks can be lessened by avoiding foods rich in cholesterol, e.g. eggs, reducing the intake of foods containing high amounts of saturated fatty acids such as animal fats, and replacing these with vegetable oils and fish that are rich in polyunsaturated fatty acids (see page 49). Blood LDL cholesterol levels may also be reduced by incorporating into the diet plant sterol esters or plant stanol esters, which reduce the absorption of cholesterol (see page 255). In humans, dietary cholesterol is actually a smaller contributor to LDL cholesterol levels than is dietary saturated fat. Cholesterol biosynthesis may also be inhibited by drug therapy using specific inhibitors of HMG-CoA reductase in the mevalonate pathway, e.g. lovastatin and related compounds (see page 98). Cholesterol is one of the primary sources for the semi-synthesis of medicinal steroids. It is currently available in quantity via the brains and spinal cords of cattle as a by-product of meat production. Large quantities are also extractable from lanolin, the fatty material coating sheep’s wool. This is a complex mixture of esters of long-chain fatty acids (including straight-chain, branched-chain, and hydroxy acids) with long-chain aliphatic alcohols and sterols. Cholesterol is a major sterol component. Saponification of crude lanolin gives an alcohol fraction (lanolin alcohols or wool alcohols) containing about 34% cholesterol and 38% lanosterol/dihydrolanosterol. Wool alcohols are also used as an ointment base.

252 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition side-chain, a trans-22 double bond, a feature seen in many plant sterols, but never in mammalian ones. The introduction of methyl and ethyl groups at C-24 generates a new chiral centre, and the 24-alkyl groups in campesterol, sitosterol, and stigmasterol are designated α. The predominant sterol found in fungi is ergosterol (Figure 5.81),

the side-chain, attached at C-24. These substituent carbon atoms are numbered 241 and 242 (Figure 5.74); some older publications may use 28 and 29. The widespread plant sterols campesterol and sitosterol (Figure 5.81) are respectively 24-methyl and 24-ethyl analogues of cholesterol. Stigmasterol contains additional unsaturation in the 242 241 22

H

24

H

24

S

E

R

H

H

H

H

H

HO

H H

H

HO

H

HO

stigmasterol

sitosterol

fucosterol

H H

H

H

HO

H

HO ergosterol

campesterol

Figure 5.81

Me

S

electrophilic addition; Ad C-methylation using SAM

W–M 1,2-hydride shift followed by loss of proton

H

26 25

H

E1

27

S R second electrophilic addition involving SAM

241

R 24

Ad Me

E1

e.g. lanosterol, cycloartenol E3 E2

H

allylic isomerization

NADPH

NADPH E3

E2 e.g. fucosterol allylic isomerization

e.g. campesterol NADPH

E3

dehydrogenation E4 dehydrogenation

NADPH

O2 NADPH

E3

E4 e.g. sitosterol

E1: sterol C-24 methyltransferase E2: 24-methylenesterol C-methyltransferase

Figure 5.82

22

e.g. stigmasterol

E3: Δ24 sterol reductase E4 sterol C-22 desaturase

O2 NADPH

22

e.g. ergosterol

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 253 which has a β-oriented 24-methyl, as well as a trans-22 double bond and additional 7 unsaturation. The descriptors α and β unfortunately, and also confusingly, do not relate to similar terms used with the steroid ring system, but are derived from consideration of Fischer projections for the side-chain, substituents to the left being designated α and those to the right as β. Systematic RS nomenclature is thus preferred, but note that this defines sitosterol as 24R, whilst stigmasterol, because of its extra double bond, is 24S. The majority of plant sterols have a 24α-methyl or 24α-ethyl substituent, whilst algal sterols tend to have 24β-ethyl groups, and fungi 24β-methyl groups. The most abundant sterol in brown algae (Fucus spp.; Fucaceae) is fucosterol (Figure 5.81), which demonstrates a further variant, a 24-ethylidene substituent. Such groups can have E-configurations, as in fucosterol, or the alternative Z-configuration. Sterols are found

predominantly in free alcohol form, but also as esters with long-chain fatty acids (e.g. palmitic, oleic, linoleic, and α-linolenic acids), as glycosides, and as glycosides acylated with fatty acids. These sterols, termed phytosterols, are structural components of membranes in plants, algae, and fungi and affect the permeability of these membranes. They also appear to play a role in cell proliferation. The source of the extra methyl or ethyl side-chain carbons in both cases is SAM, and to achieve alkylation, the side-chain must have a 24 double bond, i.e. the side-chains as seen in lanosterol and cycloartenol. The precise mechanisms involved have been found to vary according to organism, but some of the demonstrated sequences are given in Figure 5.82. Methylation of the 24 double bond at C-24 via SAM yields a carbocation which undergoes a hydride shift and loss of a proton from C-241 to generate the 24-methylene side-chain. This can be

24

C-24 alkylation

H

HO

E1 plants, algae

H

loss of C-4α methyl

H

H

E2 HO

cycloartenol

HO

H 24-methylenecycloartanol

H cycloeucalenol

opening of cyclopropane

further side-chain alkylation H HO

H

H citrostadienol (24-ethylidenelophenol)

loss of C-4 methyl

HO

H

H avenasterol

H

E6

H

E4

7

H

E9

loss of C-4 methyl

H

24-ethyl sterols HO

HO

14 8

obtusifoliol

H

24-methyl sterols

H

E1: sterol 24-C-methyltransferase E2, E8, E9: sterol 4α-methyl oxidase (sterol C-4 demethylase) E3: cycloeucalenol cycloisomerase (cycloeucalenol-obtusifoliol isomerase) E4: sterol C-14 demethylase

Figure 5.83

E5

gramisterol (24-methylenelophenol)

E8

H HO

H E7

loss of C-14α methyl and allylic isomerization (migration of Δ8 to Δ7)

E3

episterol E5: sterol Δ14-reductase E6: sterol Δ8-Δ7-isomerase (cholestenol Δ-isomerase) E7: 24-methylenesterol C-methyltransferase

254 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition 24

14

demethylations at C-14 and C-4

C-24 alkylation H

yeast

4

HO

E1

HO

H

H

H

zymosterol

lanosterol C-24 alkylation fungi

HO

fecosterol demethylations at C-4

E1

E2 E3 E4 E5

ring B and side-chain modifications

C-14 demethylation H

H HO

H eburicol (24-methylenedihydrolanosterol) E1: sterol Δ24-methyltransferase (Erg6) E2: C-8 sterol isomerase (Erg2) E3: C-5 sterol desaturase (Erg3)

HO

H

HO

H 4,4-dimethylfecosterol

ergosterol

E4: C-22 sterol desaturase (Erg5) E5: C-24(28) sterol reductase (Erg24)

Figure 5.84 reduced to a 24-methyl either directly or after allylic isomerization. Alternatively, the 24-methylene derivative acts as substrate for a second methylation step with SAM, producing a carbocation. Discharge of this cation by proton loss produces a 24-ethylidene side-chain, and reduction or isomerization/reduction gives a 24-ethyl group. The trans-22 double bond is introduced only after alkylation at C-24 is completed. No stereochemistry is intended in Figure 5.82. It is apparent that stereochemistries in the 24-methyl, 24-ethyl, and 24-ethylidene derivatives could be controlled by the reduction processes or by proton loss as appropriate. It is more plausible for different stereochemistries in the 24-methyl and 24-ethyl side-chains to arise from reduction of different double bonds, rather than reduction of the same double bond in two different ways. In practice, other mechanisms involving a 25(26)-double bond are also found to operate. The substrates for alkylation are found to be cycloartenol in plants and algae, and lanosterol in fungi. The second methylation step in plants and algae usually involves gramisterol (24-methylenelophenol; Figure 5.83). This indicates that the processes of side-chain alkylation and the steroid skeleton modifications, i.e. loss of methyl groups, opening of the cyclopropane ring, and migration of double bond, tend to run concurrently rather than sequentially. Accordingly, the range of plant and algal sterol

derivatives includes products containing side-chain alkylation, that retain one or more skeletal methyl groups, and perhaps possess a cyclopropane ring, as well as those more abundant examples such as sitosterol and stigmasterol based on a cholesterol-type skeleton. Most fungal sterols originate from lanosterol, so less variety is encountered. The pathway from lanosterol to ergosterol proceeds via zymosterol in yeast, but via eburicol (24-methylenedihydrolanosterol) in filamentous fungi; both pathways lead through fecosterol (Figure 5.84). Thus, in yeast, there is initially the same sequence of demethylations as in mammals, but then zymosterol undergoes side-chain alkylation. In fungi, side-chain alkylation occurs before the demethylations. The pathways converge at fecosterol, and ergosterol is produced by further ring B and side-chain modifications. Some useful antifungal agents, e.g. ketoconazole and miconazole, are specific inhibitors of the 14α-demethylases in fungi, but have minimal effect on cholesterol biosynthesis in humans; there is a degree of selectivity towards fungal over mammalian enzymes. Inability to synthesize the essential sterol components of their membranes proves fatal for fungi. Similarly, 14-demethylation in plants proceeds via obtusifoliol (Figure 5.83) and plants are unaffected by azole derivatives developed as agricultural fungicides.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 255 The antifungal effect of polyene antibiotics, such as amphotericin and nystatin, depends on their ability to bind strongly to ergosterol in fungal membranes and not to cholesterol in mammalian cells (see page 81). Sitosterol and stigmasterol (Figure 5.81) are produced commercially from soya beans (Glycine max; Leguminosae/Fabaceae) as raw materials for the semi-synthesis

of medicinal steroids (see pages 282, 294) [Box 5.13]. For many years, only stigmasterol was utilized, since the 22 double bond allowed chemical degradation of the side-chain to be effected with ease. The utilization of sitosterol was not realistic until microbiological processes for removal of the saturated side-chain became available.

Box 5.13 Soya Bean Sterols Soya beans or soybeans (Glycine max; Leguminosae/Fabaceae) are grown extensively in the United States, China, Japan, and Malaysia as a food plant. They are used as a vegetable, and provide a high protein flour, an important edible oil (Table 3.1), and an acceptable non-dairy soybean milk. The flour is increasingly used as a meat substitute. Soy sauce is obtained from fermented soybeans and is an indispensible ingredient in Chinese cookery. The seeds also contain substantial amounts (about 0.2%) of sterols. These include stigmasterol (about 20%), sitosterol (about 50%), and campesterol (about 20%) (Figure 5.81), the first two of which are used for the semi-synthesis of medicinal steroids. In the seed, about 40% of the sterol content is in the free form, the remainder being combined in the form of glycosides or as esters with fatty acids. The oil is usually solvent extracted from the dried flaked seed using hexane. The sterols can be isolated from the oil after basic hydrolysis as a by-product of soap manufacture, and form part of the unsaponifiable matter. The efficacy of dietary plant sterols in reducing cholesterol levels in laboratory animals has been known for many years. This has more recently led to the introduction of plant sterol esters as food additives, particularly in margarines and dairy products, as an aid to reducing blood levels of low density lipoprotein (LDL) cholesterol, known to be a contributory factor in atherosclerosis and the incidence of heart attacks (see page 251). Plant sterol esters are usually obtained by esterifying sitosterol from soya beans with fatty acids to produce a fat-soluble product. Regular consumption of this material (recommended 1.3 g per day) is shown to reduce blood LDL cholesterol levels by 10–15%. The plant sterols are more hydrophobic than cholesterol and have a higher affinity for micelles involved in fat digestion, effectively decreasing intestinal cholesterol absorption. The plant sterols themselves are poorly absorbed from the gastrointestinal tract. Of course, the average diet will naturally include small amounts of plant sterol esters. Related materials used in a similar way are plant stanol esters. Stanols are obtained by hydrogenation of plant sterols, and will consist mainly of sitostanol (from sitosterol and stigmasterol) and campestanol (from campesterol) (Figure 5.85); these are then esterified with fatty acids. Regular consumption of plant stanol esters (recommended 3.4 g per day) is shown to reduce blood LDL cholesterol levels by an average of 14%. Much of the material used in preparation of plant stanol esters originates from tall oil, a by-product of the wood pulping industry. This contains campesterol, sitosterol, and also sitostanol. The stanols are usually transesterified with rapeseed oil, which is rich in unsaturated fatty acids (see page 47). Stanols tend to be more effective in reducing cholesterol levels than sterols. It has proved possible to engineer food plants such as rapeseed and soya to produce the reduced sterols, i.e. stanols, so that the chemical reduction steps may be rendered unnecessary. This has been achieved by incorporating a Streptomyces-derived 3-hydroxysteroid oxidase gene into the plant host. This enzyme is an FAD-dependent

H H HO

Figure 5.85

H

H H

H HO

sitostanol

H

H H

H HO

campestanol

H

H stigmastanol

256 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.13 (continued) bifunctional enzyme that catalyses oxidation and isomerization in ring A of cholesterol and other steroids (compare page 264); additional reductive steps are presumably achieved by endogenous plant enzymes (Figure 5.86). Genetically modified soya seeds contained sitostanol, stigmastanol, and campestanol (Figure 5.85), with up to 80% of each normal sterol content being replaced by the reduced compound. Side-chain double bonds were not reduced.

catalysed by introduced bacterial enzyme H

H

H

E1

HO

H

H

E1

O

H

H

H

O

O

E1: 3-hydroxysteroid oxidase

H H HO

H

H

catalysed by endogenous plant enzymes

Figure 5.86

Fusidic acid (Figure 5.87), an antibacterial agent from Fusidium coccineum [Box 5.14], has no additional side-chain alkylation, but has lost one C-4 methyl and undergone hydroxylation and oxidation of a side-chain methyl. The stereochemistry in fusidic acid is not typical of most steroids, and ring B adopts a boat conformation; the chair–boat–chair–chair molecular shape is comparable to the protosteryl cation (Figure 5.63, page 238). Its relationship to the protosteryl cation is shown in Figure 5.87, and its formation probably

involves initial proton loss, which thus prevents the normal carbocation-mediated migrations. Vitamin D Vitamin D3 (colecalciferol, cholecalciferol) [Box 5.15] is a sterol metabolite formed photochemically in animals from 7-dehydrocholesterol by the sun’s irradiation of the skin (Figure 5.88). 7-Dehydrocholesterol is the immediate 5,7 diene precursor of cholesterol (see

CO2H

O H

O

H –

H

H

HO

OAc

O

H+

H HO

H

H

H HO

HO

H

H

protosteryl cation

fusidic acid H

demethylation CO2H HO

H

OH fusidic acid

Figure 5.87

O H

O

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 257 this may be considered as an extended version of an allylic isomerization, with intramolecular transfer of the proton rather than employing an external source

absorption of light energy by p-electron system allows electrocyclic ring opening

hν

H

H H

1,7-hydrogen shift H

H

H

H

HO

HO 7-dehydrocholesterol OH

OH

OH

O2 NADPH

H

rotation about C–C single bond

precholecalciferol (previtamin D3)

O2 NADPH

H

E2 kidney

9 8

E1 liver

5

10

4

HO

OH

HO

HO calcidiol (25-hydroxyvitamin D3)

calcitriol (1α,25-dihydroxyvitamin D3)

H 7

6

3

1 2

colecalciferol (cholecalciferol) (vitamin D3)

E1: cholecalciferol 25-monooxygenase E2: calcidiol 1-monooxygenase

9 10

H

hν

H

H

H

H

HO ergosterol OH

Figure 5.88

pre-ergocalciferol (previtamin D2)

HO

ergocalciferol (vitamin D2)

258 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.14 Fusidic Acid Fusidic acid (Figure 5.87) is a steroidal antibiotic produced by cultures of the fungus Acremonium fusidioides (formerly Fusidium coccineum). It has also been isolated from several Cephalosporium species. Fusidic acid and its salts are narrow-spectrum antibiotics active against Gram-positive bacteria, especially Staphylococcus. It is primarily used, as its salt sodium fusidate, in infections caused by penicillin-resistant Staphylococcus species, especially in osteomyelitis, since fusidic acid concentrates in bone. It is usually administered in combination with another antibiotic to minimize development of resistance. Fusidic acid reversibly inhibits bacterial protein biosynthesis at the translocation step by binding to the larger subunit of the ribosome (see page 422).

page 250), and a photochemical reaction allows ring opening to precholecalciferol. A thermal 1,7-hydrogen shift follows to give colecalciferol (vitamin D3 ). Vitamin D3 is manufactured photosynthetically by the same sequence. Vitamin D2 (ergocalciferol) is formed from ergosterol in exactly the same way, and is found naturally in plants and yeasts. Large amounts are produced semi-synthetically by the sequence shown in Figure 5.88, using ergosterol from yeast (Saccharomyces cerevisiae). Vitamin D3 is not itself the active form of the vitamin; in the body it is hydroxylated first to calcidiol and then to calcitriol (Figure 5.88). Colecalciferol and calcitriol have also been found in several plant species, especially members of the Solanaceae family.

Systematic nomenclature of vitamin D derivatives utilizes the obvious relationship to steroids, and the term seco (ring-opened) is incorporated into the root name (compare secologanin as a ring-opened analogue of loganin, page 207). The numbering system for steroids is also retained, and vitamin D3 becomes a derivative of 9,10-secocholestane, namely (5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-3β-ol, ‘9,10’ indicating the site of ring cleavage. Note that it is necessary to indicate the configuration of two of the double bonds. The β-configuration for the 3-hydroxyl and the α-configuration for the 1-hydroxy in calcitriol are potentially confusing until one appreciates that the bottom ring has been turned over in the cholesterol–vitamin D relationship.

Box 5.15 Vitamin D Vitamin D3 (colecalciferol, cholecalciferol; Figure 5.88) is the main form of the fat-soluble vitamin D found in animals, though vitamin D2 (ergocalciferol; Figure 5.88) is a constituent of plants and yeasts. Vitamin D3 is obtained in the diet from liver and dairy products such as butter, cream, and milk, whilst large amounts can be found in oily fish and fish liver oils, e.g. cod liver oil and halibut liver oil (Table 3.1, page 46). A major source for normal requirements is the casual exposure of the skin to sunlight, when the sterol 7-dehydrocholesterol is converted into colecalciferol by UV irradiation. With a proper diet, and sufficient exposure to sunshine, vitamin D deficency should not occur. Vitamin D deficiency leads to rickets, an inability to calcify the collagen matrix of growing bone, and is characterized by a lack of rigidity in the bones, particularly in children. In adults, osteoporosis may occur. In most countries, foods such as milk and cereals are usually fortified with vitamin D3 , obtained commercially by UV irradiation of 7-dehydrocholesterol which is produced in quantity by semi-synthesis from cholesterol. Vitamin D2 has a similar activity in humans and is manufactured by UV irradiation of yeast, thereby transforming the ergosterol content. Other compounds with vitamin D activity have also been produced: vitamin D4 from 22,23-dihydroergosterol, vitamin D5 from 7-dehydrositosterol, vitamin D6 from 7-dehydrostigmasterol, and vitamin D7 from 7-dehydrocampesterol. Vitamin D1 was an early preparation later shown to be a mixture of vitamin D2 and a photochemical by-product lumisterol. Lumisterol (9β,10α-ergosterol) is formed by recyclization of pre-ergocalciferol (Figure 5.88), giving the ergosterol stereoisomer. Vitamin D is unstable to heat, light, and air. Vitamin D3 is not itself the active form of the vitamin, and in the body it is hydroxylated firstly to 25-hydroxyvitamin D3 (calcidiol; Figure 5.88) by an enzyme in the liver, and then to 1α,25-dihydroxyvitamin D3 (calcitriol) by a kidney enzyme; both enzymes are cytochrome P-450 systems. Calcitriol is then transported to the bones, intestine, and other organs. It stimulates calcium absorption from the intestine, reabsorption from the kidney, and mobilization from bone. Calcitriol and other analogues, e.g. alfacalcidol, dihydrotachysterol, and paricalcitol (Figure 5.89), are available for use where chronic vitamin D deficiency is due to liver or kidney malfunction. The long-term use of calcitriol and alfacalcidol (1α-hydroxyvitamin D3 ) in the treatment of osteoporosis may lead to toxic effects arising from elevated serum calcium levels.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 259

Box 5.15 (continued) Vitamin D is also known to have other physiological functions, including a role in immune suppression, hormone secretion, and the differentiation of both normal and malignant cells. Vitamin D derivatives, including calcipotriol, tacalcitol, and maxacalcitol (Figure 5.89), are widely used in the topical treatment of psoriasis, to inhibit the cell proliferation characteristic of this condition. In maxacalcitol, the side-chain modification (22-methylene replaced by an ether link) appears to retain the non-calcaemic action whilst reducing calcaemic activity, the latter probably because of more rapid oxidative metabolism in the liver. Vitamin D2 is also employed as a rodenticide. High doses are toxic to rats and mice, since the vitamin causes fatal hypercalcaemia.

OH

H

HO

H

OH alfacalcidol

H

HO

HO dihydrotachysterol

OH

OH paricalcitol

OH O OH

H

HO

OH calcipotriol

H

HO

OH tacalcitol

H

HO

OH maxacalcitol

Figure 5.89

Steroidal Saponins Steroidal saponins have similar biological properties to the triterpenoid saponins, e.g. surfactant and haemolytic activities (see page 242), but are less widely distributed in nature. They are found in many monocot families, especially the Dioscoreaceae (e.g. Dioscorea), the Agavaceae (e.g. Agave, Yucca) and the Liliaceae (e.g. Smilax, Trillium). Their sapogenins are C27 sterols in which the side-chain of cholesterol has undergone modification to produce either a spiroketal (spirostane saponins), e.g. dioscin, or a hemiketal (furostane saponins), e.g. protodioscin (Figure 5.90). As described below, the furostanes feature

in the biosynthetic sequence to the spirostanes. All the steroidal saponins have the same configuration at the centre C-22, but stereoisomers at C-25 exist, e.g. yamogenin (Figure 5.91), and often mixtures of the C-25 stereoisomers co-occur in the same plant. Sugars are found at position 3, with a second glycoside function at C-26 in the furostanes. The sugar moiety at position 3 typically contains fewer monosaccharide units than are found with triterpenoid saponins; one to three monosaccharide units are most common. In general, the more sugar residues there are attached, the greater is the haemolytic activity. The sugar at C-26 in furostanes is usually glucose.

260 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O H D-Glc

HO HO L-Rha

H

22

H β1→ α1→4 L-Rha D-Glc O α1→2 L-Rha

OH

O

H

diosgenin

O HO L-Rha

OH

H

3

α1→2 O OH HO

OGlc 26

H H

O

HO

25

22

O

H

OH O β1→ O

α1→4 O

26

H

H

protodioscin furostane

dioscin spirostane

Characteristic features of steroidal saponins: spirostanes: • spiroketal at C-22 • common configuration at C-22 • sugar residues on 3β-hydroxyl

furostanes: • hemiketal at C-22 • common configuration at C-22 • sugar residues on 3β-hydroxyl and 26-hydroxyl

Figure 5.90

O H

dioscin

22

H+

H H

3

25

H

O

H

H+

O

25

O

H

H

protodioscin

H

H

HO

H

HO yamogenin

diosgenin

O

O

O

H

12

H

O

H

O HO

H H

H

H H

H HO

diosgenin

H

H hecogenin

Figure 5.91 Acid hydrolysis of either dioscin or protodioscin liberates the aglycone diosgenin (Figure 5.91); the hydrolytic conversion of protodioscin into diosgenin is analogous to the biosynthetic sequence. The three-dimensional shape of diosgenin is indicated in Figure 5.91. The spiroketal function is derived from the cholesterol side-chain by a series of oxygenation reactions, hydroxylating one of the terminal methyl groups and at C-16, and then producing a ketone function at C-22 (Figure 5.92). This proposed intermediate is transformed into the hemiketal and then the spiroketal. The

chirality at C-22 is fixed by the stereospecificity in the formation of the ketal, whilst the different possible stereochemistries at C-25 are dictated by whether C-26 or C-27 is hydroxylated in the earlier step. Enzymic glycosylation at the 3-hydroxyl of spirostane sapogenins has been reported, but knowledge of other steps at the enzymic level is lacking. Furostane derivatives, e.g. protodioscin (Figure 5.90), can co-occur with spirostanes, and undoubtedly represent glycosylation of the intermediate hemiketal at the 26-hydroxyl. These compounds are readily hydrolysed and then

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 261 22

25

26

O

O

H OH

27

H H

O OH

O

16

OH

H hydroxylations at C-26, then C-16

HO cholesterol

OH

H

nucleophilic attack of hydroxyl onto ketone gives hemiketal

H

H

O

H

O H

3

H

22

H H

HO

H

UDPGlc OGlc OH

25

H

H+

O

H

H

ketal (spiroketal) formation from hemiketal

H

saponins

OH

H O

UDPsugars

OH

H

HO

O H

chemical hydrolysis allows spontaneous formation of spiroketal

H furostane

diosgenin/yamogenin

Figure 5.92 spontaneously cyclize to the spiroketal. Allowing homogenized fresh plant tissues to stand and autolyse through the action of endogenous glycosidase enzymes not only achieves cyclization of such open-chain saponins, but also can hydrolyse off the sugar units at C-3, thus yielding the aglycone or sapogenin. This is a standard approach employed in commercial production of steroidal sapogenins, which are important starting materials for the semi-synthesis of steroidal drugs [Box 5.16]. Diosgenin is the principal example and is obtained from Mexican yams (Dioscorea spp.; Dioscoreaceae). Fenugreek (Trigonella foenum-graecum; Leguminosae/Fabaceae) is another potentially useful commercial source of diosgenin. Sisal (Agave sisalana; Agavaceae) is also used commercially, yielding hecogenin (Figure 5.91), a 12-keto derivative with trans-fused A/B rings, the result of reduction of the 5 double bond. Undoubtedly related to the furostans and spirostans is the sterol glycoside OSW-1 (Figure 5.93) isolated from the bulbs of Ornithogalum saundersiae (Liliaceae). This material has shown quite remarkable cytotoxicity, inhibiting the growth of various tumour cells, and being 10–100 times more potent than some clinical anticancer agents. It appears to damage the mitichondrial membrane in

O 22

OH 17

H H

16

←α1 O AcO

H

O

O

HO O

HO HO D-Xyl OSW-1

L-Ara

O

OH

β1→3

O OMe

Figure 5.93 cancer cells, so its mechanism of action is also different to current anticancer agents. In addition, it shows little toxicity towards normal cells. The aglycone part of OSW-1 is structurally similar to early intermediates in the spirostanol pathway, in that it possesses the 22-keto and 16-hydroxyl functions, with an additional hydroxyl at C-17. 17α-Hydroxylation is characteristic of corticosteroids (see page 278) and the oestrogen/androgen biosynthetic pathway (see page 291).

262 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.16 Dioscorea About 600 species of Dioscorea (Dioscoreaceae) are known, and a number of these are cultivated for their large starchy tubers, commonly called yams, which are an important food crop in many parts of the world. Important edible species are Dioscorea alata and D. esculenta (Southeast Asia), D. rotundata and D. cayenensis (West Africa) and D. trifida (America). A number of species accumulate quite high levels of saponins in their tubers, which make them bitter and inedible, but these provide suitable sources of steroidal material for drug manufacture. Dioscorea spp. are herbaceous, climbing, vine-like plants, the tuber being totally buried, or sometimes protruding from the ground. Tubers weigh anything up to 5 kg, but in some species the tubers have been recorded to reach weights as high as 40–50 kg. Drug material is obtained from both wild and cultivated plants, with plants collected from the wild having been exploited considerably more than cultivated ones. Commercial cultivation is less economic, requiring a 4–5-year growing period and some form of support for the climbing stems. Much of the world’s production has come from Mexico, where tubers from D. composita (barbasco), D. mexicana, and D. floribunda, mainly harvested from wild plants, are utilized. The saponin content of the tubers varies, usually increasing as tubers become older. Typically, tubers of D. composita may contain 4–6% total saponins, and D. floribunda 6–8%. Other important sources of Dioscorea used commercially now include India (D. deltoidea), South Africa (D. sylvatica) and China (D. collettii, D. pathaica, and D. nipponica). Sapogenins are isolated by chopping the tubers, allowing them to ferment for several days, and then completing the hydrolysis of saponins by heating with aqueous acid. The sapogenins can then be solvent extracted. The principal sapogenin in the species given above is diosgenin (Figure 5.91), with small quantities of the 25β-epimer yamogenin (Figure 5.91). Demand for diosgenin for pharmaceuticals is huge, equivalent to 10 000 t of Dioscorea tuber per annum, and it is estimated that about 60% of all steroidal drugs are derived from diosgenin. Powdered Dioscorea (wild yam) root or extract is also marketed to treat the symptoms of menopause as an alternative to hormone replacement therapy (HRT; see page 294). Although there is a belief that this increases levels of progesterone, which is then used as a biosynthetic precursor of other hormones, there is little definitive evidence that diosgenin is metabolized in the human body to progesterone, and any beneficial effects may arise from diosgenin itself.

Fenugreek The seeds of fenugreek (Trigonella foenum-graecum; Leguminosae/Fabaceae) are an important spice material, and are ingredients in curries and other dishes. The plant is an annual and is grown widely, especially in India, both as a spice and as a forage crop. Seeds can yield, after hydrolysis, 1–2% of sapogenins, principally diosgenin (Figure 5.91) and yamogenin (Figure 5.91). Although yields are considerably lower than from Dioscorea, the ease of cultivation of fenugreek and its rapid growth make the plant a potentially viable crop for steroid production in temperate countries. Field trials of selected high-yielding strains have been conducted.

Sisal Sisal (Agave sisalana; Agavaceae) has long been cultivated for fibre production, being the source of sisal hemp, used for making ropes, sacking, and matting. The plant is a large, rosette-forming succulent with long, tough, spine-tipped leaves containing the very strong fibres. The main area of sisal cultivation is East Africa (Tanzania, Kenya), with smaller plantations in other parts of the world. The sapogenin hecogenin (Figure 5.91) was initially produced from the leaf waste (0.6–1.3% hecogenin) after the fibres had been stripped out. The leaf waste was concentrated, allowed to ferment for several days, and then treated with steam under pressure to complete hydrolysis of the saponins. Filtration then produced a material containing about 12% hecogenin, plus other sapogenins. This was refined further in the pharmaceutical industry. Other sapogenins present include tigogenin and neotigogenin (Figure 5.94). As the demand for natural fibres declined due to the availability of synthetics, so did the supply of sisal waste and, thus, hecogenin. In due course, hecogenin became a more valuable commodity than sisal, and efforts were directed specifically towards hecogenin production. This has resulted in the cultivation of Agave hybrids with much improved hecogenin content. The highest levels (2.5%) of hecogenin recorded have been found in a Mexican species Agave sobria var roseana. The fermented sap of several species of Mexican Agave, especially Agave tequilana, provides the alcoholic beverage pulque. Distillation of the fermented sap produces tequila.

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 263

Box 5.16 (continued)

O

O

H

H O

H A BH HO

H

H

A BH HO

H

O

H

H

H

H

A/B cis, smilagenin A/B trans, tigogenin

A/B cis, sarsasapogenin A/B trans, neotigogenin

Figure 5.94

H N H

OH O

HO HO D-Glc

D-Gal OH OH

O OH β1→3

HO L-Rha

H H

H

O

O

HO

22

O

H

O β1→ O

α1→2

25

solasodine OH solasonine

HO HO D-Xyl

25

D-Glc OH O β1→4 O HO O O OH OH β1→3 OH O β1→ O O HO HO O OH HO OH β1→2 D-Gal D-Glc

NH

H

22

O

H

H H

H

H tomatidine

tomatine

Figure 5.95 Some steroidal alkaloids are nitrogen analogues of steroidal saponins and display similar properties, such as surface activity and haemolytic activity, but these compounds are toxic when ingested. These types of compound, e.g. solasonine (Figure 5.95; aglycone solasodine), are found in many plants of the genus Solanum (Solanaceae), and such plants must thus be regarded as

potentially toxic. In contrast to the oxygen analogues, all compounds have the same stereochemistry at C-25 (methyl always equatorial), whilst isomers at C-22 do exist, e.g. tomatine (Figure 5.95; aglycone tomatidine) from tomato (Lycopersicon esculente; Solanaceae). The nitrogen atom is introduced by a transamination reaction, typically employing an amino acid as donor (see page

264 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition oxidation of hydroxyl to C=O H NAD H 5

HO NADPH

H

reduction E1 of Δ5,6

keto–enol tautomerism

H

E2

6

H

+

E3

O

H H

E3

HO

H H

enol–keto tautomerism H O

H

H

H

4

5

E4

NADPH H

reduction of C=O

reduction of Δ4,5 H

NADPH H HO

H HO

H A/B trans E1: Δ5-steroid 5α-reductase E2: Δ5-3β-hydroxysteroid dehydrogenase E3: Δ5-3-ketosteroid isomerase

H A/B cis

H E5 O

H

E4: Δ4-3-ketosteroid 5β-reductase E5: 3β-hydroxysteroid dehydrogenase

Figure 5.96 409). Since the production of medicinal steroids from steroidal saponins requires preliminary degradation to remove the ring systems containing the original cholesterol side-chain, it is immaterial whether these rings contain oxygen or nitrogen. Thus, plants rich in solasodine or tomatidine can also be employed for commercial steroid production (see page 410). Smilagenin and sarsasapogenin (Figure 5.94) found in sarsaparilla (Smilax spp.; Liliaceae/Smilacaceae) are reduced forms of diosgenin and yamogenin respectively. These contain cis-fused A/B rings, whilst the corresponding trans-fused systems are present in tigogenin and neotigogenin (Figure 5.94) found in Digitalis purpurea along with cardioactive glycosides (see page 269). All four stereoisomers are derived from cholesterol, and the stereochemistry of the A/B ring fusion appears to be

controlled by the nature of the substrate being reduced. Enzymic reduction of the isolated 5 double bond yields the trans-fused system, whereas reduction of a 4 double bond (1,4-addition to a conjugated ketone) gives the alternative cis-fused system (Figure 5.96). Accordingly, to obtain the A/B cis fusion, the 5 unsaturation of cholesterol is changed to 4 by oxidation of the 3-hydroxyl and allylic isomerization to the conjugated 4-ene-3-one system, and this is followed by reduction of both functional groups (Figure 5.96) (compare biosynthesis of progesterone, page 267). The sarsaparilla saponins are not present in sufficient quantities to be commercially important for steroid production, but quite large amounts of sarsasapogenin can be extracted from the seeds of Yucca brevifolia (Agavaceae) [Box 5.17].

Box 5.17 Sarsaparilla Sarsaparilla consists of the dried roots of various Smilax species (Liliaceae/Smilacaceae), including S. aristolochiaefolia, S. regelii, and S. febrifuga, known respectively as Mexican, Honduran, and Ecuadorian sarsaparilla. The plants are woody climbers indigenous to Central America. Sarsaparilla has a history of use in the treatment of syphilis, rheumatism, and skin diseases, but is now mainly employed as a flavouring in the manufacture of non-alcoholic drinks. It has some potential as a raw material for the semi-synthesis of medicinal steroids, being a source of sarsasapogenin and smilagenin (Figure 5.94). The roots contain 1.8–2.4% steroidal saponins, including parillin (Figure 5.97).

Yucca Yucca brevifolia (Agavaceae) has been explored as a potential source of sarsasapogenin for steroid production, especially at times when market prices of diosgenin from Dioscorea became prohibitively expensive. The plant grows extensively in the

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 265

Box 5.17 (continued)

O HO HO

OH O

O

HO L-Rha

HO OH HO

H

O β1→ O

HO

O

HO

O

H D-Glc

OH

D-Glc α1→4 O

H

β1→6

OH O

D-Glc

O OH β1→2

H

H

H

sarsasapogenin

parillin Figure 5.97 Mojave Desert in California, and high levels of sarsasapogenin (8–13%) are present in the seeds. This means the plants can be harvested regularly without damage. The subsequent stabilization of Dioscorea prices in the 1970s stopped any further commercial exploitation.

Cardioactive Glycosides Many of the plants known to contain cardiac or cardiotonic glycosides have long been used as arrow poisons (e.g. Strophanthus) or as heart drugs (e.g. Digitalis). They are used medicinally to strengthen a weakened heart and allow it to function more efficiently, though the dosage must be controlled very carefully, since the therapeutic dose is so close to the toxic dose. The cardioactive effects of Digitalis were discovered as a result of its application in the treatment of dropsy, an accumulation of water in the body tissues. Digitalis alleviated dropsy indirectly by its effect on the heart, improving the blood supply to the kidneys and so removing excess fluid. The therapeutic action of cardioactive glycosides depends on the structure of the aglycone, and on the type and number of sugar units attached. Two types of aglycone are recognized, cardenolides (e.g. digitoxigenin from D. purpurea), which are C23 compounds, and bufadienolides (e.g. hellebrigenin from Helleborus niger), which are C24 structures (Figure 5.98). Stereochemistry is very important for activity, and these compounds have cis fusions for both the A/B and C/D ring junctions, 3β- and 14β-hydroxyl groups with the glycoside function at C-3, and an α,β-unsaturated lactone grouping at C-17β. This lactone ring is five-membered

in the cardenolides and six-membered in the bufadienolides. The hellebrigenin structure shows two other modifications not found in the basic steroid skeleton, namely a hydroxyl at the bridgehead carbon C-5 and a formyl group at C-10, being an oxidized form of the normal methyl. The three-dimensional shape of digitoxigenin is shown in Figure 5.98. These basic structures arise biosynthetically by metabolism of cholesterol, in which the side-chain is cleaved to a two-carbon acetyl group, followed by incorporation of either two carbonatoms for cardenolides or three carbonatoms for bufadienolides (Figure 5.99). Shortening of the cholesterol side-chain is accomplished by stepwise hydroxylation at C-22 and then C-20, then cleavage of the C-20/22 bond to give pregnenolone, a sequence catalysed by the ‘side-chain cleaving enzyme’. Pregnenolone is then oxidized in ring A to give progesterone (Figure 5.99). This can be reduced to give the cis-fused A/B system as in 5β-pregnan-3,20-dione (compare Figure 5.96) which is the substrate for 14β-hydroxylation, i.e. inverting the stereochemistry at this centre. Inversion is atypical for hydroxylation by monooxygenases, which are found to hydroxylate with retention of configuration. Whatever the mechanism of this hydroxylation, no 8 or 15 double-bond intermediates are involved. Hydroxylation in the side-chain at C-21 follows. The lactone ring is

266 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition O O

O 23 22

21 20

H

3

HO

22

20 17

OHC

14

10

OH

H

23

21

17

H

24

O

3

HO

digitoxigenin cardenolide

5

H H

14

OH O

OH

Characteristic features of cardiac glycosides: • cis-fused A/B and C/D rings • 14β-hydroxyl • unsaturated lactone at C-17β • sugar residues on 3β-hydroxyl

O

H

hellebrigenin bufadienolide

OH

H

H H HO

digitoxigenin

Figure 5.98 created at this stage. An intermediate malonate ester is involved, and ring formation probably occurs via the aldol addition process shown in Figure 5.100 to give the cardenolide digitoxigenin, the carboxyl carbon of the malonate ester being lost by decarboxylation during the process (compare the role of malonate in the acetate pathway). Digitoxigenin is the precursor of digoxigenin and gitoxigenin by specific hydroxylations (Figure 5.99). Oxaloacetate is the source of the extra carbon atoms in bufadienolides; a similar esterification/aldol reaction sequence may be proposed (Figure 5.100). This would produce bufalin (Figure 5.99), a bufadienolide structure found in the skin of toad (Bufo spp.), from which this class of compound was originally isolated and has subsequently taken the general name. Note that, in the subsequent formation of hellebrigenin (Figure 5.99), hydroxylation at C-5 occurs with the expected retention of stereochemistry, not with inversion as seen at C-14. The fundamental pharmacological activity of the cardioactive glycosides resides in the aglycone portion, but is considerably modified by the nature of the sugar at C-3. This increases water solubility and binding to heart muscle. The sugar unit may have one to four monosaccharides; many (e.g. d-digitoxose and d-digitalose; Figure 5.101) are unique to this group of compounds. About 20 different sugars have been characterized, and with the exception of d-glucose, they are 6-deoxy- (e.g. l-rhamnose, d-digitalose) or 2,6-dideoxy- (e.g. d-digitoxose, d-cymarose) hexoses, some of which are also 3-methyl ethers (e.g. d-digitalose

and d-cymarose; Figure 5.101). In plants, cardiac glycosides are confined to the angiosperms, but are found in both monocotyledons and dicotyledons. The cardenolides are more common, and the plant families the Apocynaceae (e.g. Strophanthus), Liliaceae (e.g. Convallaria), and Scrophulariaceae (e.g. Digitalis) yield medicinal agents [Box 5.18]. The rarer bufadienolides are found in some members of the Liliaceae (e.g. Urginea) [Box 5.18] and Ranunculaceae (e.g. Helleborus), as well as in toads. Monarch butterflies and their larvae are known to accumulate in their bodies a range of cardenolides which they ingest from their food plant, the common milkweed (Asclepias syriaca; Asclepiadaceae). This makes them unpalatable to predators such as birds. Endogenous Digitalis-like compounds have also been detected, albeit in very small quantities, in mammalian tissues. Ouabain (see below, Figure 5.106), first isolated from the bark of the African ouabio tree (Acokanthera ouabio; Apocynaceae), is now known to occur naturally in blood plasma, adrenal glands, and the hypothalamus of mammals; it is a major component in the seeds of Strophanthus gratus [Box 5.18]. 19-Norbufalin (Figure 5.101) is found in the lens of human eyes, at higher levels if these are cataract-afflicted, and it is believed to regulate ATPase activity under some physiological and pathological conditions. Spirostane saponins are known to co-occur with cardenolide glycosides in D. purpurea (see page 264), indicating the plant is able to synthesize structures in which A/B and C/D ring fusions may be trans–trans or

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 267

20

oxidative cleavage between hydroxyls, perhaps via peroxide

stepwise hydroxylation via 22-hydroxycholesterol

22

H

HO

H

H

H

E1

HO

O

OH

H

O2 NADPH

H

H

E1

HO

pregnenolone Δ4,5

reduction of gives O cis-fused A/B rings H H

3

H

E3

H

E2

H

H

H H

H

E2

HO

O

progesterone

O

HO

H O

NAD+

keto–enol tautomerism

enol–keto tautomerism

reduction of 3-ketone E4 to 3b-hydroxyl

H

E2

O H

NADPH

H 5β-pregnan-3,20-dione NADPH

H

HO

cholesterol

O

OH

O

O

H

O2 NADPH

H

O

14b-hydroxylation with inversion of stereochemistry

H

H HO

O side-chain 21-hydroxylation

H

14

H

OH

21

OH

H HO

H

3β-hydroxy5β-pregnan-20-one

H

3β,14β-dihydroxy5β-pregnan-20-one

OH

H 3β,14β,21-trihydroxy5β-pregnan-20-one HO2C

malonyl-CoA

O

see Figure 5.100 E5

O

O

H

H OH

H HO

H

12b-hydroxylation O

OH

H

5b-hydroxylation and C-19 oxidation

bufalin

digitoxigenin 16b-hydroxylation O

O

O O

O

HO 12

H H HO

H digoxigenin

H OH

H HO

16

OHC

OH

H gitoxigenin

E1: cholesterol monooxygenase (side-chain cleaving) (side-chain cleaving enzyme) Ε2: Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase

Figure 5.99

19

OH

5

HO

H H

SCoA

O oxaloacetyl-CoA

O

H

HO

O

OH

OH hellebrigenin

E3: progesterone 5β-reductase E4: 3β-hydroxysteroid dehydrogenase E5: 21-hydroxypregnane 21-O-malonyltransferase

268 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

O

O

CO2H CoAS malonyl-CoA

OH

aldol reaction with concomitant decarboxylation; compare malonyl-CoA as chain extender in acetate pathway O O CO2H

O

O

O

O

O

O

H

dehydration favoured by formation of conjugated system O

O

– H 2O

– CO2

H

O

O

OH ester formation cardenolide

malonate ester O CoAS OH

O

CO2H O

O oxaloacetyl-CoA O

aldol reaction with concomitant decarboxylation O O – CO2

O O

O

O

O

CO2H

O

O H

H

O O OH

ester formation oxaloacetate ester O O

dehydration favoured by formation of conjugated system

– H 2O

1,4-elimination favouring formation of conjugated dienone

O

O

O

O

OH reduction

O

H

bufadienolide

Figure 5.100 cis–cis. Other variants are possible, in that compounds such as digipurpurogenin II (Figure 5.102) have also been found in D. purpurea. This compound has the C/D ring fusion cis, with a 14β-hydroxyl group, characteristic of the cardiac glycosides, yet the A/B rings are typical of more common sterols. The glycoside P57A3 of this compound found in the South African succulents Hoodia pilifera and H. gordonii is attracting considerable interest as an appetite suppressant. Indigenous people have used the plants as a substitute for food and water, and appetite-suppressant properties were subsequently confirmed, leading to the isolation of P57A3 and its glycoside (a cymaroside). A Hoodia herbal appetite suppressant has been patented. Though these compounds are pregnane derivatives, they bear a strong relationship to cardenolides via the 12β- and 14β-oxygenation, and the chain of 2,6-dideoxy sugar units.

HO

6

6

O

HO

O OH

2

MeO

OH 3

OH D-digitoxose

OH O

D-digitalose O

6

O

HO 2

OH

OMe D-cymarose

H

H H

HO

Figure 5.101

OH

H 19-norbufalin

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 269 O O

HO MeO

O β1→4 O

D-thevatose

H

O β1→4 O OMe D-cymarose

O

O β1→ O

H

OH

OMe D-cymarose

12-O-tigloyldigipurpurogenin II

P57A3

Figure 5.102

Box 5.18 Digitalis purpurea Digitalis leaf consists of the dried leaf of the red foxglove Digitalis purpurea (Scrophulariaceae). The plant is a biennial herb, common in Europe and North America, which forms a low rosette of leaves in the first year and its characteristic spike of purple (occasionally white) bell-shaped flowers in the second year. It is potentially very toxic, but the leaf is unlikely to be ingested by humans. D. purpurea is cultivated for drug production, principally in Europe, the first year leaves being harvested then rapidly dried at 60◦ C as soon as possible after collection. This procedure is necessary to inactivate hydrolytic enzymes which would hydrolyse glycoside linkages in the cardioactive glycosides, giving rise to less active derivatives. Even so, some partial hydrolysis does occur. Excess heat may also cause dehydration in the aglycone to biologically inactive 14 -anhydro compounds. Because of the pronounced cardiac effects of digitalis, the variability in the cardiac glycoside content, and also differences in the range of structures present due to the effects of enzymic hydrolysis, the crude leaf drug is usually assayed biologically rather than chemically. Prepared digitalis is a biologically standardized preparation of powdered leaf, its activity being assessed on cardiac muscle of guinea pig or pigeon and compared against a standard preparation. It may be diluted to the required activity by mixing in powdered digitalis of lower potency, or inactive materials such as lucerne (Medicago sativa) or grass. The crude drug is hardly ever used now, having been replaced by the pure isolated glycosides. The cardioactive glycoside content of D. purpurea leaf is 0.15–0.4%, consisting of about 30 different structures. The major components are based on the aglycones digitoxigenin, gitoxigenin, and gitaloxigenin (Figure 5.103), the latter being a formate ester. The glycosides comprise two series of compounds, those with a tetrasaccharide glucose–(digitoxose)3 – unit and those with a trisaccharide (digitoxose)3 – unit. The latter group (the secondary glycosides) is produced by partial hydrolysis from the former group (the primary glycosides) during drying by the enzymic action of a β-glucosidase which removes the terminal glucose. Thus, the principal glycosides in the fresh leaves, namely purpureaglycoside A and purpureaglycoside B (Figure 5.103), are partially converted into digitoxin and gitoxin respectively (Figure 5.103), which normally predominate in the dried leaf. These transformations are indicated schematically in Figure 5.104. In the fresh leaf, purpureaglycoside A can constitute about 50% of the glycoside mixture, whilst in the dried leaf the amounts could be negligible if the plant material is old or poorly stored. The gitaloxigenin-based glycosides are relatively unstable, and the formyl group on the aglycone is readily lost by hydrolysis. Other minor glycosides are present, but neither the fresh nor dried leaf contains any significant quantities of the free aglycones. Glycosides of the gitoxigenin series are less active than the corresponding members of the digitoxigenin-derived series. Digitoxin is the only compound routinely used as a drug, and it is employed in congestive heart failure and treatment of cardiac arrhymias, particularly atrial fibrillation.

Digitalis lanata Digitalis lanata (Scrophulariaceae), the Grecian foxglove, is a perennial or biennial herb from southern and central Europe. It differs in appearance from the red foxglove by its long narrow smoother leaves and its smaller flowers of a yellow–brown colour. It is cultivated in Europe, the United States, and South America and is harvested and dried in a similar manner to D. purpurea. It has not featured as a crude drug, but is used exclusively for the isolation of individual cardiac glycosides, principally digoxin and lanatoside C (Figure 5.105).

270 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.18 (continued) O

O OH O β1→4 O OH

HO HO

O β1→4 O OH

D-glucose

OH

D-digitoxose

H

O β1→4 O

O β1→ O OH

D-digitoxose

D-digitoxose

H

R OH

H R = H, digitoxigenin R = OH, gitoxigenin R = O2CH, gitaloxigenin

R = H, digitoxin R = OH, gitoxin R = O2CH, gitaloxin R = H, purpureaglycoside A R = OH, purpureaglycoside B R = O2CH, glucogitaloxin OH O β1→4 O OH

HO HO

O β1→4 O O

D-glucose

O β1→4 O OH

O 3-acetylD-digitoxose

O

O

D-digitoxose

H O β1→ O OH D-digitoxose

H

16

R

OH

H R = H, digitoxigenin R = OH, gitoxigenin R = O2CH, gitaloxigenin

R = H, acetyldigitoxin R = OH, acetylgitoxin R = O2CH, acetylgitaloxin R = H, lanatoside A R = OH, lanatoside B R = O2CH, lanatoside E

O

O

R1 HO HO

OH O

12

β1→4

OH O D-glucose

H

O β1→4 O

O

O 3-acetylD-digitoxose

O β1→4 O OH D-digitoxose

O β1→ O OH D-digitoxose

H

16

OH

H 1

R = OH, R2 = H, digoxigenin R1 = OH, R2 = OH, diginatigenin

R1 = OH, R2 = H, acetyldigoxin R1 = OH, R2 = OH, acetyldiginatin R1 = OH, R2 = H, lanatoside C R1 = OH, R2 = OH, lanatoside D

Figure 5.103

R2

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 271

Box 5.18 (continued) purpureaglycoside A

lanatoside A

Glc–Dig–Dig–Dig–digitoxigenin

Glc–Dig–Dig–Dig–digitoxigenin Ac

– Glc digitoxin

– Glc

acetyldigitoxin – Ac

Dig–Dig–Dig–digitoxigenin

Dig–Dig–Dig–digitoxigenin Ac

purpureaglycoside B

lanatoside B

Glc–Dig–Dig–Dig–gitoxigenin

Glc–Dig–Dig–Dig–gitoxigenin Ac

– Glc gitoxin

– Glc

acetylgitoxin – Ac

Dig–Dig–Dig–gitoxigenin

Dig–Dig–Dig–gitoxigenin Ac

desacetyl-lanatoside C

lanatoside C – Ac Glc–Dig–Dig–Dig–digoxigenin

Glc–Dig–Dig–Dig–digoxigenin

Ac

– Glc digoxin

– Glc

acetyldigoxin – Ac

Dig–Dig–Dig–digoxigenin

lanatoside D Dig–Dig–Dig–digoxigenin Glc–Dig–Dig–Dig–diginatigenin

Ac

Ac glucogitaloxin

lanatoside E

Glc–Dig–Dig–Dig–gitaloxigenin

Glc–Dig–Dig–Dig–gitaloxigenin Ac

– Glc

– Glc

acetyldiginatin Dig–Dig–Dig–diginatigenin Ac

gitaloxin

Dig–Dig–Dig–gitaloxigenin Ac

Figure 5.104

– Ac

acetylgitaloxin – Ac

Dig–Dig–Dig–gitaloxigenin

– Glc

diginatin Dig–Dig–Dig–diginatigenin

272 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.18 (continued) O

O

OH H

O

R1O

O

O

H

O

O

OR2

O OH 1

2

OH

H

OH

R = R = H, digoxin R1 = Me, R2 = H, medigoxin (metildigoxin) R1 = Glc, R2 = Ac, lanatoside C R1 = Glc, R2 = H, deslanoside (desacetyl-lanatoside C)

Figure 5.105 The total cardenolide content of up to 1% is two to three times that found in D. purpurea. The main constituents resemble those of D. purpurea, but contain an acetyl ester function on the third digitoxose, that furthest from the aglycone. This acetyl group makes the compounds easier to isolate from the plant material and they crystallize more readily. Drying of the leaf is similarly accompanied by some partial hydrolysis of the original fresh leaf constituents through enzymic action, and both the terminal glucose and the acetyl group may be hydrolysed off, extending the range of compounds isolated. The D. lanata cardiac glycosides are based on five aglycones: digitoxigenin, gitoxigenin and gitaloxigenin, as found in D. purpurea, plus digoxigenin and diginatigenin (Figure 5.103), which do not occur in D. purpurea. The primary glycosides containing the acetylated tetrasaccharide unit glucose–acetyldigitoxose–(digitoxose)2 – are called lanatosides. Lanatosides A and C (Figure 5.103) constitute the major components in the fresh leaf (about 50–70%) and are based on the aglycones digitoxigenin and digoxigenin respectively. Lanatosides B, D, and E (Figure 5.103) are minor components derived from gitoxigenin, diginatigenin, and gitaloxigenin respectively. Enzymic hydrolysis of the lanatosides generally involves loss of the terminal glucose prior to removal of the acetyl function, so that compounds like acetyldigitoxin and acetyldigoxin, as well as digitoxin and digoxin, are present in the dried leaf as decomposition products from lanatosides A and C respectively. These transformations are also indicated in simplified form in Figure 5.104. Digoxin (Figure 5.105) has a rapid action and is more quickly eliminated from the body than digitoxin; therefore, it is the most widely used of the cardioactive glycosides. It is more hydrophilic than digitoxin, binds less strongly to plasma proteins, and is mainly eliminated by the kidneys, whereas digitoxin is metabolized more slowly by the liver. Digoxin is used in congestive heart failure and atrial fibrillation. Lanatoside C and deslanoside (desacetyl-lanatoside C) (Figure 5.105) have also been employed, though not to the same extent. They have very rapid action and are suited for treatment of cardiac emergencies by injection. The semi-synthetic derivative medigoxin or metildigoxin (methyl replacing the glucose in lanatoside C; Figure 5.105) has also been available, being more active through better bioavailability. The cardioactive glycosides increase the force of contractions in the heart, thus increasing cardiac output and allowing more rest between contractions. The primary effect on the heart appears to be inhibition of the ion transport activity of the enzyme Na+ /K+ -ATPase in the cell membranes of heart muscle, specifically inhibiting the Na+ pump, thereby raising the intracellular Na+ concentration. The resultant decrease in the Na+ gradient across the cell membrane reduces the energy available for transport of Ca2+ out of the cell, leads to an increase in intracellular Ca2+ concentration, and provides the positive ionotropic effect and increased force of contractions. The improved blood circulation also tends to improve kidney function, leading to diuresis and loss of oedema fluid often associated with heart disease. However, the diuretic effect, historically important in the treatment of dropsy, is more safely controlled by other diuretic drugs. To treat congestive heart failure, an initial loading dose of the cardioactive glycoside is followed by regular maintenance doses, the amounts administered depending on drug bioavailability and subsequent metabolism or excretion. Because of the extreme toxicity associated with these compounds (the therapeutic level is 50–60% of the toxic dose; a typical daily dose is only about 1 mg), dosage must be controlled very carefully. Bioavailability has sometimes proved erratic and can vary between different manufacturers’ formulations, so patients should not be provided with different preparations during their treatment. Individual patients also excrete the glycosides or metabolize them by hydrolysis to the aglycone at different rates, and ideally these processes should be monitored. Levels of the drug in blood plasma can be measured quite rapidly by radioimmunoassay using a specific antibody. A preparation of digoxin-specific antibody fragments derived from sheep is available both for assay

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 273

Box 5.18 (continued) and also as a means of reversing life-threatening digoxin overdose. It has also successfully reversed digitoxin overdose, thus demonstrating a somewhat broader specificity. The value of digoxin treatment for heart failure where the heartbeat remains regular has recently been called into question. It still remains a recognized treatment for atrial fibrillation. Many other species of Digitalis, e.g. D. dubia, D. ferruginea, D. grandiflora, D. lutea, D. mertonensis, D. nervosa, D. subalpina, and D. thaspi contain cardioactive glycosides in their leaves, and some have been evaluated and cultivated for drug use. Gene engineering may be able to increase the amount of cardioactive glycosides obtainable. Thus, transgenic D. minor plants expressing an additional gene for the mevalonate pathway enzyme HMG-CoA reductase from Arabidopsis thaliana have shown increased sterol and cardenolide production.

Strophanthus Strophanthus comprises the dried ripe seeds of Strophanthus komb´e or S. gratus (Apocynaceae), which are tall vines from equatorial Africa. S. komb´e has a history of use by African tribes as an arrow poison, and the seeds contain 5–10% cardenolides, a mixture known as K-strophanthin. This has little drug use today, though was formerly used medicinally as a cardiac stimulant. The main glycoside (about 80%) is K-strophanthoside (Figure 5.106) with smaller amounts of K-strophanthin-β and cymarin, related to K-strophanthoside as shown. These are derivatives of the aglycone strophanthidin. S. gratus contains 4–8% of ouabain (G-strophanthin; Figure 5.106), the rhamnoside of ouabigenin. Ouabigenin is rather unusual in having additional hydroxylation O

O OH O

HO HO

HO

α1→4 O HO

D-glucose

OHC

OH O β1→4 O OH

O

β1→

D-glucose

H

O OMe D-cymarose

H OH

OH strophanthidin

cymarin K-strophanthin-β K-strophanthoside O HO HO HO 1

α1→ O

10

HO

H

OHC OH

HO

ouabain (G-strophanthin)

OH

OH

O

HO ouabigenin

H H

α1→ O

OH

OH

L-rhamnose

O

H

O

HO

Figure 5.106

11

O

OH

L-rhamnose

strophanthidin

convallatoxin

O

274 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition

Box 5.18 (continued) at 1β and 11α, as well as a hydroxymethyl at C-10. Ouabain is a stable, crystalline material which is often employed as the biological standard in assays for cardiac activity. It is a potent cardiac glycoside, acts quickly, but wears off rapidly. It is very polar, with rapid renal elimination and must be injected because it is so poorly absorbed orally. It has been used for emergency treatment in cases of acute heart failure.

Convallaria The dried roots and tops of lily of the valley Convallaria majalis (Liliaceae/Convallariaceae) contain cardioactive glycosides (0.2–0.3%) and in the past have been used in some European countries rather than digitalis. The effects are similar, but the drug is less cumulative. This plant is widely cultivated as an ornamental, particularly for its intensely perfumed small white flowers, and must be considered potentially toxic. The major glycoside (40–50%) is convallatoxin (Figure 5.106), the rhamnoside of strophanthidin.

Squill Squill (white squill) consists of the dried sliced bulbs of the white variety of Urginea maritima (formerly Scilla maritima; also known as Drimia maritima) (Liliaceae/Hyacinthaceae) which grows on seashores around the Mediterranean. The plant contains bufadienolides (up to 4%), principally scillaren A and proscillaridin A (Figure 5.107). The aglycone of scillaren A is scillarenin, which is unusual in containing a 4 double bond and, thus, lacks the cis A/B ring fusion found in the majority of cardiac glycosides. Squill is not usually used for its cardiac properties, as the glycosides have a short duration of action. Instead, squill is employed for its expectorant action in preparations such as Gee’s linctus. Large doses cause vomiting and a digitalis-like action on the heart. Red squill is a variety of U. maritima which contains an anthocyanin pigment (see page 170) and bufadienolides which are different from those of the white squill. The main glycosides are glucoscilliroside and scilliroside (Figure 5.107), glucosides of scillirosidin. This chemical variety should not be present in medicinal squill, and has mainly been employed as a rodenticide. Rodents lack a vomiting reflex and are poisoned by the cardiac effects, whilst vomiting will occur in other animals and humans due to the emetic properties of the drug. The use of red squill as a rodenticide is now considered inhumane.

Toxic Plants: Cardioactive Glycosides Many plants containing cardioactive glycosides are widely grown as ornamentals and must be considered toxic and treated with due care and respect. These include Digitalis species, Convallaria majalis, Helleborus species, and oleander (Nerium oleander; Apocynaceae). O O

O O

H H α1→ O OH β1→4 O HO O HO O HO OH OH D-glucose L-rhamnose

OH HO HO

scillarenin

OH

OH O β1→4 O HO OH D-glucose

OH β1→ O O OH D-glucose scilliroside

proscillaridin A scillaren A

Figure 5.107

glucoscilliroside

H OAc scillirosidin

OH

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 275 O CO2H

HO

HO

N H

12

H 3

5

HO

H

H

OH

H

HO

H OH

H

sodium glycocholate

cholic acid H

H2N

H

SO3H O

taurine HO

H

HO H

HO

H

Na

H

H2N CO2H glycine

H 7

CO2

N H

CO2H

Characteristic features of bile acids: • C24 cholane skeleton • cis-fusion of A/B rings • C5-carboxylic acid side-chain • 3α- and 7α-hydroxyls

Na

H

OH cholic acid

SO3

H HO

H

H OH sodium taurocholate

Figure 5.108 Bile Acids The bile acids are C24 steroidal acids, e.g. cholic acid (Figure 5.108), which occur in salt form in bile, secreted into the gut to emulsify fats and encourage digestion [Box 5.19]. The carboxyl group is typically bound via an amide linkage to glycine (about 75%) or taurine (about 25%), e.g. cholic acid is found as sodium glycocholate and sodium taurocholate. Conjugation with glycine or taurine increases the water solubility of bile salts under physiological conditions. Taurine (2-aminoethanesulphonic acid) was first isolated from ox bile, but is now known to be widely distributed in animal tissues. Metabolism to bile acids is the principal way in which mammals degrade cholesterol absorbed from the diet. Cholesterol is extremely hydrophobic; its removal is dependent upon increasing hydrophilicity, achieved by the introduction of several polar groups into the molecule. The cis fusion of rings A and B confers a curvature to the steroidal skeleton, and the polar hydroxyl groups are all positioned on the lower α face, contrasting with the non-polar upper β face. Because of this ambiphilicity, they can form micelles and act as detergents. The bile acids are formed in the liver from cholesterol by a sequence which also removes three carbon atoms from the side-chain (Figure 5.109). This is achieved by

initial oxidation of one of the side-chain methyl groups to an acid, followed by a β-oxidation sequence as seen with fatty acids (see Figure 2.11), removing the three-carbon unit as propionyl-CoA. Other essential features of the molecule are introduced earlier. The A/B ring system is cis-fused, and this is achieved by reduction of a 4 rather than a 5 double bond (see page 264). Migration of the double bond is accomplished via the 3-ketone; when this is reduced back to a hydroxyl, the configuration at C-3 is changed to 3α. The pathway to chenodeoxycholic acid (Figure 5.110) is essentially the same, though diverges early on by omitting the 12α-hydroxylation step. Alternative pathways (‘acidic’ pathways) to bile acids are known, though the one described (the ‘classical’ or ‘neutral’ pathway) predominates. These other pathways are characterized by initial 27-hydroxylation in the side-chain, and side-chain modifications tend to precede modifications in the sterol nucleus. Both cholic acid and chenodeoxycholic acid are formed in the liver, stored in the gall bladder, and released into the intestine; they are termed primary bile acids. However, the 7α-hydroxyl functions of these compounds can be removed by intestinal microflora, so that mammalian bile also contains deoxycholic acid and lithocholic acid (Figure 5.110), which are termed secondary bile acids. The bile salts are then usually reabsorbed and stored in

276 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition 7α-hydroxylation; oxidation of 3-hydroxyl leads to Δ4-3-ketone, then 12α-hydroxylation

O

reduction of 4,5-double bond gives cis-fused rings; stereospecific reduction of carbonyl generates 3α-hydroxyl HO

O

HO

12

O2 NAD+ O2 NADPH NADPH

H H

3

7

HO

H

E1

E2

E3

O

H H

H

O

H

NADPH NADH E4

OH

H

E5 HO

H OH oxidation of side-chain methyl O2 to carboxyl NADPH

H

cholesterol E6

O activation of acid to coenzyme A ester

O COSCoA

HO H H HO

H

H HO

H

shortening of side-chain; b-oxidation removes E8 propionyl-CoA

H

glycine

H HO

H HO

H

E9

OH

H

H OH

H

CO2H

HO H H

HO

H

H OH

cholic acid

choloyl-CoA E1: E2: E3: E4: E5:

H

H

E10 taurine

E7

COSCoA

HO

glycocholate taurocholate

H OH

CO2H

HO

ATP HSCoA

H

b-oxidation OH

COSCoA

HO

cholesterol 7α-hydroxylase (cholesterol 7α-monooxygenase) cholest-5-ene-3β,7α-diol 3β-dehydrogenase 7α-hydroxycholest-4-ene-3-one 12α-hydroxylase Δ4-3-ketosteroid 5β-reductase 3α-hydroxysteroid dehydrogenase

E6: cholestanetriol 26-monooxygenase E7: cholestanate-CoA ligase E8: propanoyl-CoA C-acyltransferase E9: cholate-CoA ligase E10: bile acid-CoA: amino acid N-acyltransferase

Figure 5.109

CO2H

R

intestinal microflora

H H HO

H

R = OH, cholic acid R = H, chenodeoxycholic acid

Figure 5.110

CO2H

H

H OH

CO2H

R

H HO

H H

H R = OH, deoxycholic acid R = H, lithocholic acid

H HO

H 7

H

OH

ursodeoxycholic acid

The Mevalonate and Methylerythritol Phosphate Pathways: Terpenoids and Steroids 277 the gall bladder, although they are also excreted as the body’s main means of eliminating excess cholesterol. Inability to remove cholesterol by bile acid synthesis and excretion may contribute to atherosclerosis and gallstone disease; gallstones often contain more than 70% of cholesterol (see page 251). 7α-Hydroxylation of cholesterol is

the critical and rate-limiting step. This is catalysed by the cytochrome P-450-dependent monooxygenase cholesterol 7α-hydroxylase, and any deficiency of this enzyme leads to high levels of total serum and LDL cholesterol (see page 251).

Box 5.19 Bile Acids Bile acids are obtained by purification from fresh ox bile taken from beef carcasses as a by-product of the meat trade. Bile acids are still important as starting materials for the semi-synthesis of other medicinal steroids, being a cheap and readily accessible raw material. The 7-epimer of chenodeoxycholic acid, ursodeoxycholic acid (ursodiol; Figure 5.110), is a minor secondary bile acid in humans, but a major component in bear bile. It is produced semi-synthetically from cholic acid or chenodeoxycholic acid and used medicinally to dissolve cholesterol gallstones as an alternative to surgery. By suppressing synthesis of both cholesterol and cholic acid, they contribute to removal of biliary cholesterol and, consequently, a gradual dissolution of gallstones which can have formed due to supersaturation. Partial or complete dissolution requires treatment over a period of many months, and is not effective for radio-opaque gallstones, which contain appreciable levels of calcium salts. Anion-exchange resins such as colestyramine (cholestyramine) and colestipol are used as cholesterol-lowering drugs to bind bile acids and prevent their reabsorption. This promotes hepatic conversion of cholesterol into bile acids, thus increasing breakdown of LDL cholesterol, and is of value in treating high-risk coronary patients.

Adrenocortical Hormones/Corticosteroids A large number of steroid hormones have been isolated and characterized from the adrenal glands. Since they are produced by the adrenal cortex, the outer part of the adrenal glands near the kidneys, they are termed adrenocortical hormones or corticosteroids [Box 5.20]. They contain a pregnane C21 skeleton and fall into two main activity groups, the glucocorticoids and the mineralocorticoids, although it is difficult to separate entirely the two types of activity in one molecule. Glucocorticoids are concerned with the synthesis of carbohydrate from protein and the deposition of glycogen in the liver. They also play an important role in inflammatory processes. Mineralocorticoids are concerned with the control of electrolyte balance, active compounds promoting the retention of Na+ and Cl− , and the excretion of K+ . Examples of natural glucocorticoids include hydrocortisone (cortisol) and corticosterone, whilst aldosterone and deoxycorticosterone (cortexone) typify mineralocorticoids (Figure 5.111). Deoxycorticosterone has also been found in plants. Some common features of these molecules are the β-CO.CH2 OH side-chain at C-17 and, frequently, an α-hydroxy also at this position. Ring A usually contains a 4 -3-keto functionality. The 11β-hydroxy is essential for glucocorticoid activity. In aldosterone, the principal mineralocorticoid hormone, the methyl group (C-18) has been oxidized to an aldehyde, and this is able

to react with the 11β-hydroxyl, so that aldosterone exists predominantly in the hemiacetal form. This essentially eliminates the glucocorticoid activity. The corticosteroids are produced from cholesterol via pregnenolone and progesterone. This involves side-chain cleavage as seen in the biosynthesis of cardioactive glycosides (see page 267), and the same sequence of reactions is operative. From progesterone, the formation of deoxycorticosterone, corticosterone, and hydrocortisone (cortisol) (Figure 5.112) requires only a series of hydroxylation steps, catalysed by cytochrome P-450-dependent hydroxylases with NADPH and O2 cofactors. Thus, positions 17, 21, and 11 may be hydroxylated, and the exact order can in fact vary from that shown in Figure 5.112, according to species. It can be seen that production of hydrocortisone from cholesterol actually utilizes cytochrome P-450-dependent enzymes in four of the five steps. The further oxidation of C-18 to an aldehyde via the alcohol allows formation of aldosterone from corticosterone, again involving a P-450 system. Semi-Synthesis of Corticosteroids The medicinal use of corticosteroids was stimulated by reports of the dramatic effects of cortisone (Figure 5.113) on patients suffering from rheumatoid arthritis in the late 1940s and early 1950s. The cortisone employed

278 Medicinal Natural Products: A Biosynthetic Approach. 3rd Edition OH

HO

17

11

OH

O OH

O HO

H 3

H

4

H H

H

O

H

O hydrocortisone (cortisol)

corticosterone

Characteristic features of glucocorticoids: • C21 pregnane skeleton • 17β-CO.CH2OH side-chain • 11β-hydroxyl • Δ4-3-keto (usually) • 17α-hydroxyl (usually) OH O

OHC

HO

O

4

O

O

17

H 3

OH

OH HO

H

H

H H

O

H

H

O

O aldosterone

H

H

aldosterone (hemiacetal form)

deoxycorticosterone (cortexone)

Characteristic features of mineralocorticoids: • C21 pregnane skeleton • 17β-CO.CH2OH side-chain • Δ4-3-keto (usually) Figure 5.111 was isolated from the adrenal glands of cattle, and later was produced semi-synthetically by a laborious process from deoxycholic acid (see page 276) isolated from ox bile, a sequence necessitating over 30 chemical steps. In due course, it was shown that cortisone itself was not the active agent; it was reduced in the liver to hydrocortisone as the active agent (Figure 5.113). Two dehydrogenase enzymes regulate hydrocortisone levels, both of them acting in a unidirectional sense. One of these activates cortisone by reduction, and though prevalent in the liver is found in a wide range of tissues. The other enzyme is found predominantly in mineralocorticoid target tissues, e.g. kidney and colon, where it protects receptors against excess hydrocortisone by converting it to inactive cortisone. Increased demand for cortisone and hydrocortisone (cortisol) led to exploitation of alternative raw materials, particularly plant sterols and saponins. A major difficulty in any semi-synthetic conversion was

the need to provide the 11β-hydroxyl group, which was essential for glucocorticoid activity. Sarmentogenin (Figure 5.114) had been identified as a natural 11-hydroxy cardenolide in Strophanthus sarmentosus, but it was soon appreciated that the amounts present in the seeds, and the limited quantity of plant material available, would not allow commercial exploitation of this compound. As an altern